Chiral LC/MS/MS Analysis with Polysaccharide-Based Stationary

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Pharmaceutical Compounds and NeutralStereoisomeric Stationary PhasesforBasic with Polysaccharide-based Chiral LC/MS/MSAnalysis Phenomenex, Inc.,411MadridAve.,Torrance, CA 90501USA(www.phenomenex.com) Liming Peng,TivadarFarkasandSwapanaJayapaian

PO87820611_L_2 Introduction

The hyphenation of the resolving power of chiral HPLC compatible with atmospheric pressure ionization (API) with the sensitivity of MS detection is highly desired MS ion sources. Developing rapid chiral LC separations in drug metabolism and pharmacokinetic studies of compatible with MS ionization interfaces while preserving stereoisomers in the drug discovery process. Derivatives both chromatographic resolution and sensitivity in MS of polysaccharides are the most widely used chiral detection has proven to be a great challenge to analytical stationary phases due to their wide chiral recognition scientists. ability, high loading capacity, and durability. As normal phase is favorable for the principal mechanism of chiral We present the results of a systematic feasibility study recognition – hydrogen bonding interaction – the majority of using polysaccharide-based chiral stationary phases of chiral separations with polysaccharide phases are (CSPs) coupled with API-MS/MS detection for the performed in normal phase using hexane and alcohol analysis of various pharmaceutical racemates in reversed modifiers as mobile phase components. However phase (RP) elution mode. these mobile phases are highly flammable and are not Experimental Conditions

HPLC System: HP 1100 series Flow Rate: 0.2 mL/min (www.agilent.com) Injection Volume: 5 µL 0.2 – 1.0 µg/mL of racemates Pump: G1312A (Binary Pump) Concentration: Columns: Lux 3 µm Cellulose-1 Autosampler: G1329A ALS 150 x 2.0 mm MS Detector: API 3000 LC/MS/MS with ESI Lux 3 µm Cellulose-2 (TurboIonSpray®) 150 x 2.0 mm (www. Appliedbiosystems.com) Lux 3 µm Amylose -2 150 x 2.0 mm Gemini®-NX 3 µm C18 50 x 2.0 mm TurbolonSpray - ESI, Positive Ion Mobile Phases: 1. Acetonitrile/Methanol : 5 mM Mode; MRM; heater gas flow 5000 NH4HCO3 2. Acetonitrile/Methanol: 5 mM cc/min; heater temperature 400 ˚C. NH4HCO3 + 0.025 % DEA* 3. Acetonitrile/Methanol: 5 mM CH3COONH4 * DEA - Diethylamine Figure 2. LC/MS/MS Responses of Representative Racemates 0.1 % Formic acid/Acetonitrile

4.E+07 5 mM Ammonium Bircarbonate/Acetonitrile

1.E+07

9.E+06 4.E+07 0.1 % Formic acid/CH3CN 8.E+06 5 mM NH4HCO3/CH3CN

7.E+06

6.E+06 3.E+07 esponses 5.E+06

4.E+06

3.E+06 LC/MS/MS r

3.E+07 2.E+06

1.E+06 esponses

0.E+00 Conc.: 200 ng/mL 2.E+07 Column: Gemini-NX 3 µm Ketamine Isoxsuprine Nifenolol Nisoldipine rimipramine T Norketamine 50 x 2.0 mm 2.E+07 LC/MS/MS r 1.E+07

5.E+06

0.E+00

Ketamine Nifenolol Isoxsuprine Nisoldipine rimipramine T Norketamine Figure 3. Effect of Modifier and Additive on Enantiorecognition

XIC of +MRM (1 pair): 225.0/165.2 amu from Sample ... Max. 6.5e5 cps. XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 1.8e5 cps.

8.83 15.18 Intensity, cps 8.42 Intensity, cps 1.8e5 16.09 6.0e5 Nifenolol 225.0/165.2 1.5e5 Trimipramine 295.4/100.0 5.0e5 Lux Cellulose-2 Lux Cellulose-2 4.0e5 60:40/MeOH: 5 mM NH4HCO3 50:50/CH3CN: 5 mM NH4HCO3 1.0e5 3.0e5

2.0e5 5.0e4 1.0e5 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 16 18 20 22 24 Time, min Time, min XIC of +MRM (1 pair): 225.0/165.2 amu from Sample ... Max. 1.6e4 cps. XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 1.5e5 cps.

Intensity, cps 8.15 8.77 Intensity, cps 22.71 24.21 1.5e5 1.5e4 Decreased signal Nifenolol 225.0/165.2 Trimipramine 295.4/100.0 24.44 Lux Cellulose-2 Lux Cellulose-2 1.0e5 1.0e4 60:40//MeOH: 5 mM NH4HCO3 +0.025 % DEA 45:55/CH3CN: 5 mM NH4HCO3

5000.0 5.0e4 Broader peaks & delayed RT

0.0 0.0 2 4 6 8 10 12 14 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Time, min Time, min XIC of +MRM (1 pair): 225.0/165.2 amu from Sample... Max. 3881.0 cps. XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 4.8e4 cps.

Intensity, cps 25.77 Intensity, cps 18.69 19.92 3881 4.8e4

29.12 Trimipramine 295.4/100.0 Decreased signal Nifenolol 225.0/165.2 4.0e4 3000 Lux Cellulose-2 Lux Cellulose-2 50:50/CH3CN: 5 mM NH4HCO3 +0.025 % DEA 45:55/MeOH: 5 mM NH4HCO3 + 0.025 % DEA 3.0e4 2000 35 min 2.0e4 1000 1.0e4

0 0.0 5 10 15 20 25 30 2 4 6 8 10 12 14 16 18 20 22 24 Time, min Time, min Figure 4. Effects of Temperature and Additive on Enantiorecognition

XIC of +MRM (4 pairs): 180.0/124.1 amu from Sample... Max. 4.4e5 cps. XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 8.8e4 cps.

Intensity, cps 4.66 Intensity, cps 5.83 4.4e5 8.8e4 6.52 4.0e5 5.52 8.0e4 Lorazepam 321.4/275.1 3.0e5 Adrenaline 180.0/124.1 6.0e4 Lux Cellulose-2 Lux Cellulose-2 80:20/MeOH: 5 mM NH4HCO3 2.0e5 50:50/MeOH: 5 mM NH4HCO3 4.0e4 @ ambient @ ambient 1.0e5 2.0e4

0.0 0.0 2 4 6 8 10 2 4 6 8 Time, min Time, min XIC of +MRM (4 pairs): 180.0/124.1 amu from Samplee... Max. 1.9e5 cps. XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 1.9e4 cps.

Intensity, cps 6.61 Intensity, cps 7.81 1.9e5 1.9e4 Broad peaks & delayed retention 9.03 8.50 1.5e5 1.5e4

1.0e5 Adrenaline 180.0/124.1 1.0e4 Lorazepam 321.4/275.1 Lux Cellulose-2 Lux Cellulose-2 5.0e4 50:50/MeOH: 5 mM NH4HCO3 5000.0 80:20/MeOH: 5 mM NH4HCO3 @ 5ºC @ 5ºC 2.95 0.0 0.0 2 4 6 8 10 2 4 6 8 10 Time, min Time, min XIC of +MRM (4 pairs): 180.0/124.1 amu from Sample... Max. 2.0e5 cps. XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 2313.2 cps.

Intensity, cps 4.55 Intensity, cps 5.66 2.0e5 2313 6.32 Decreased signal 5.40 2000 1.5e5 Lorazepam 321.4/275.1 Adrenaline 180.0/124.1 1500 Lux Cellulose-2 Lux Cellulose-2 80:20/MeOH: 5 mM 1.0e5 50:50/MeOH: 5 mM 1000 NH HCO +0.025 % DEA NH HCO + 0.025 % DEA 4 3 4 3 @ ambient 5.0e4 @ ambient 500

0.0 0 2 4 6 8 10 2 4 6 8 10 Time, min Time, min Figure 5a. Effect of Buffer on Chiral Resolution in RP LC/MS/MS on Lux Cellulose-1

XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 1.6e4 cps. XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 4120.0 cps.

Intensity, cps 4.30 Intensity, cps 3.45 1.6e4 3.75 4000 3.96 1.0e4 Aminoglutethimide 233.2/188.3 Aminoglutethimide 233.2/188.3 60:40/CH3CN: 5 mM NH4HCO3 2000 60:40/CH3CN: 5 mM NH4AC 2.74 0.0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 181.2/141.2 amu from Sampl... Max. 3.3e4 cps. XIC of +MRM (9 pairs): 181.2/141.2 amu from Sample... Max. 4.1e4 cps.

Intensity, cps 4.41 Intensity, cps 2.63 3.3e4 5.94 4.0e4 2.43 Orphenadrin 181.2/141.2 Orphenadrin 181.2/141.2 60:40/CH3CN: 5 mM NH4HCO3 2.0e4 60:40/CH3CN: 5 mM NH4AC

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (13 pairs): 366.1/132.2 amu from Sample... Max. 2.9e5 cps. XIC of +MRM (14 pairs): 366.1/132.2 amu from Sample... Max. 2.3e4 cps.

Intensity, cps 4.76 Intensity, cps 4.33 2.9e5 6.17 2.0e4 2.0e5 Indapamide 366.1/132.2 5.65 Indapamide 366.1/132.2 60:40/CH3CN: 5 mM NH4HCO3 60:40/CH3CN: 5 mM NH4AC 1.0e5 1.0e4 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 2.6e5 cps. XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 1.4e5 cps.

Intensity, cps 8.73 9.41 Intensity, cps 4.63 4.89 2.6e5 1.4e5 2.0e5 Motofoline 346.3/125.1 1.0e5 Motofoline 346.3/125.1 60:40/CH CN: 5 mM NH HCO 60:40/CH CN: 5 mM NH AC 1.0e5 3 4 3 5.0e4 3 4 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 370.4/252.1 amu from Sample... Max. 2.1e6 cps. XIC of +MRM (9 pairs): 370.4/252.1 amu from Sampl... Max. 2.6e5 cps.

Intensity, cps 3.86 Intensity, cps 3.44 2.0e6 2.6e5 Lansoprizole 370.4/252.1 2.0e5 Lansoprizole 370.4/252.1 1.0e6 60:40/CH3CN: 5 mM NH4HCO3 1.0e5 60:40/CH3CN: 5 mM NH4AC 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 5b. Effect of Buffer on Chiral Resolution in RP LC/MS/MS on Lux Cellulose-1

XIC of +MRM (9 pairs): 245.1/217.3 amu from Sample... Max. 2.5e4 cps. XIC of +MRM (9 pairs): 245.1/217.3 amu from Sample... Max. 8660.0 cps.

Intensity, cps 3.97 Intensity, cps 3.52 4.06 2.5e4 4.58 Zopiclone 245.1/217.3 8660 2.0e4 60:40/CH3CN: 5 mM NH4HCO3 Zopiclone 245.1/217.3 5000 60:40/CH CN: 5 mM NH AC 1.0e4 5.84 6.34 5.41 5.88 3 4 0.0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 314.3/176.2 amu from Sample... Max. 4.1e6 cps. XIC of +MRM (9 pairs): 314.3/176.2 amu from Sample... Max. 3.1e6 cps.

Intensity, cps 4.40 Intensity, cps 2.92 3.86 4.0e6 6.56 3.1e6 Reboxetine 314.3/176.2 Reboxetine 314.3/176.2 2.0e6 60:40/CH3CN: 5 mM NH4HCO3 60:40/CH3CN: 5 mM NH4AC

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 3.6e5 cps. XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 2.4e5 cps.

Intensity, cps 5.41 Intensity, cps 4.17 3.6e5 5.93 4.56 2.0e5 Nomifensine 239.3/44.1 Nomifensine 239.3/44.1 60:40/CH3CN: 5 mM NH4HCO3 1.0e5 60:40/CH3CN: 5 mM NH4AC 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 5.0e5 cps. XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 6.7e5 cps.

Intensity, cps 6.98 Intensity, cps 2.77 7.96 6.7e5 4.0e5 Mebeverine 430.4/149.1 5.0e5 Mebeverine 430.4/149.1 2.0e5 60:40/CH3CN: 5 mM NH4HCO3 60:40/CH3CN: 5 mM NH4AC 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 272.4/129.4 amu from Samp... Max. 5.1e5 cps. XIC of +MRM (14 pairs): 272.4/129.4 amu from Samp... Max. 2.3e5 cps.

Intensity, cps 6.74 7.02 Intensity, cps 6.18 6.45 5.1e5 2.0e5 4.0e5 Napropamide 272.4/129.4 Napropamide 272.4/129.4 2.0e5 60:40/CH3CN: 5 mM NH4HCO3 1.0e5 60:40/CH3CN: 5 mM NH4AC 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 6. Enantioresolution in RP LC/MS/MS on Lux Cellulose-1

XIC of +MRM (11 pairs): 293.4/248.3 amu from Sample... Max. 1.5e6 cps. XIC of -MRM (4 pairs): 420.0/289.1 amu from Sample... Max. 1.1e5 cps. Intensity, cps Intensity, cps 4.01 3.51 1.5e6 3.93 5.63 1.00e5 1.0e6 Dimethindene 293.4/248.3 Bendroﬂumethiazde 420.0/289.1 in 90:10/MeOH: 5 mM NH4HCO3 (negative mode) 5.0e5 5.00e4 in 80:20/MeOH: 5 mM NH4HCO3 0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 Time, min Time, min XIC of +MRM (11 pairs): 285.2/200.2 amu from Sample... Max. 9.5e5 cps. XIC of +MRM (3 pairs): 276.3/124.2 amu from Sample... Max. 8.3e5 cps. Intensity, cps Intensity, cps 6.49 3.33 9.5e5 8.3e5 4.09 7.87 Etozolin 285.2/200.2 Homatropine 276.3/124.2 5.0e5 5.0e5 in 90:10/MeOH: 5 mM NH4HCO3 in 80:20/MeOH: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (11 pairs): 299.4/104.2 amu from Sample... Max. 1.5e6 cps. XIC of +MRM (6 pairs): 272.3/44.1 amu from Sample ... Max. 4.4e5 cps. Intensity, cps Intensity, cps 4.43 6.82 8.39 1.5e6 7.01 4.0e5 1.0e6 Isoamarine 299.4/104.2 Nisoxetine 272.3/44.1 in 90:10/MeOH: 5 mM NH4HCO3 2.0e5 in 40:60/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (11 pairs): 265.3/208.3 amu from Sample... Max. 1.6e5 cps. XIC of +MRM (6 pairs): 264.3/58.2 amu from Sample ... Max. 1.0e6 cps. Intensity, cps Intensity, cps 6.60 5.60 6.13 1.6e5 1.00e6 8.13 1.0e5 Mianserin 265.3/208.3 Tramadol 264.3/58.2 in 90:10/MeOH: 5 mM NH4HCO3 5.00e5 in 40:60/CH3CN: 5 mM NH4HCO3

0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (11 pairs): 500.3/142.3 amu from Sample... Max. 1.6e4 cps. XIC of +MRM (3 pairs): 391.2/201.1 amu from Sample... Max. 4.1e6 cps. Intensity, cps Intensity, cps 10.67 7.98 8.59 1.6e4 12.44 4.0e6 1.0e4 Halofantrine 500.3/142.3 Meclizine 391.2/201.1 in 90:10/MeOH: 5 mM NH4HCO3 2.0e6 in 70:30/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 7a. Enantioresolution in RP LC/MS/MS on Lux Cellulose-2

XIC of +MRM (14 pairs): 246.3/98.2 amu from Sample... Max. 2.1e5 cps. XIC of +MRM (14 pairs): 299.4/104.2 amu from Sample... Max. 2.8e5 cps.

Intensity, cps 5.55 6.54 Intensity, cps 5.16 5.57 2.0e5 2.8e5 Tolperisone 246.3/98.2 2.0e5 Isoamarine 299.4/104.2 1.0e5 60:40/CH3CN: 5 mM NH4HCO3 1.0e5 60:40/CH3CN: 5 mM NH4HCO3 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 251.3/132.3 amu from Sample... Max. 9.1e4 cps. XIC of +MRM (14 pairs): 272.4/129.4 amu from Sample... Max. 4.5e5 cps.

Intensity, cps 4.77 Intensity, cps 6.63 7.15 9.1e4 4.0e5 Methaqualone 251.3/132.3 Napropamide 272.4/129.4 5.0e4 60:40/CH3CN: 5 mM NH4HCO3 2.0e5 60:40/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 259.1/84.2 amu from Sample... Max. 1.4e4 cps. XIC of +MRM (14 pairs): 328.3/120.2 amu from Sample... Max. 3021.8 cps.

Intensity, cps 3.78 Intensity, cps 2.80 1.4e4 3022 1.0e4 5.80 Thalidomide 259.1/84.2 Omeprazole 328.3/120.2 60:40/CH3CN: 5 mM NH4HCO3 5000.0 2.62 60:40/CH3CN: 5 mM NH4HCO3 0.0 0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 205.1/178.3 amu from Sample... Max. 3.8e5 cps. XIC of +MRM (14 pairs): 398.4/124.2 amu from Sample... Max. 1.0e6 cps.

Intensity, cps 4.88 Intensity, cps 6.44 3.8e5 5.44 1.00e6 7.29 Tetramisole 205.1/178.3 Diperodon 398.4/124.2 60:40/CH3CN: 5 mM NH4HCO3 5.00e5 60:40/CH3CN: 5 mM NH4HCO3

0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 5.4e5 cps. XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 2.0e4 cps.

Intensity, cps 6.85 7.34 Intensity, cps 3.61 3.96 5.4e5 2.0e4 4.0e5 Mebeverine 430.4/149.1 Aminoglutethimide 233.2/188.3 60:40/CH CN: 5 mM NH HCO 1.0e4 60:40/CH CN: 5 mM NH HCO 2.0e5 3 4 3 3 4 3 3.24 4.30 13.78 0.0 0.0 2 4 6 8 9.05 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 7b. Enantioresolution in RP LC/MS/MS on Lux Cellulose-2

XIC of +MRM (14 pairs): 246.3/98.2 amu from Sample... Max. 5.5e5 cps. XIC of +MRM (13 pairs): 299.3/104.3 amu from Sample... Max. 3.4e5 cps. Intensity, cps Intensity, cps 3.98 3.12 5.5e5 3.63 3.4e5 4.0e5 Tolperisone 246.3/98.2 Isoamarine 299.3/104.3 80:20/CH3CN: 5 mM NH4HCO3 80:20/CH CN: 5 mM NH HCO 2.0e5 3 4 3 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (13 pairs): 311.2/243.3 amu from Sample... Max. 4.1e6 cps. XIC of +MRM (13 pairs): 285.2/200.2 amu from Sample... Max. 7.5e5 cps. Intensity, cps Intensity, cps 5.42 6.08 4.15 4.0e6 7.5e5 5.69 Bifonazole 311.2/243.3 5.0e5 Etozolin 285.2/200.2 2.0e6 80:20/CH3CN: 5 mM NH4HCO3 80:20/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (13 pairs): 362.3/91.2 amu from Sample... Max. 5.1e4 cps. XIC of +MRM (13 pairs): 466.2/184.0 amu from Sample... Max. 7.5e5 cps. Intensity, cps Intensity, cps 4.56 4.26 5.1e4 6.71 7.5e5 5.19 4.0e4 Butaclamol 362.3/91.2 5.0e5 Cisapride 466.2/184.0 80:20/CH CN: 5 mM NH HCO 2.0e4 3 4 3 80:20/CH3CN: 5 mM NH4HCO3 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (13 pairs): 293.4/164.3 amu from Sample... Max. 3.7e5 cps. XIC of +MRM (13 pairs): 381.1/125.1 amu from Sample... Max. 7.3e5 cps. Intensity, cps Intensity, cps 5.39 7.39 3.7e5 7.3e5 8.67 Ambucetamid 293.4/164.3 5.0e5 Econazole 381.1/125.1 80:20/CH3CN: 5 mM NH4HCO3 7.20 80:20/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 417.1/159.1 amu from Sample... Max. 1.9e5 cps. XIC of +MRM (13 pairs): 500.3/142.3 amu from Sample... Max. 1.0e4 cps. Intensity, cps Intensity, cps 10.20 10.95 11.59 1.9e5 12.07 1.00e4 Miconaxole 417.1/159.1 Halofantrine 500.3/142.3 1.0e5 80:20/CH3CN: 5 mM NH4HCO3 5000.00 80:20/CH3CN: 5 mM NH4HCO3 12.48 0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 7c. Enantioresolution in RP LC/MS/MS on Lux Cellulose-2

XIC of +MRM (9 pairs): 314.3/176.2 amu from Sample... Max. 4.7e6 cps. XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample... Max. 4.1e5 cps. Intensity, cps Intensity, cps 4.06 4.58 4.17 4.0e5 5.12 4.0e6 Reboxetine 314.3/176.2 Nomifensine 239.3/44.1 2.0e6 60:40/CH3CN: 5 mM NH4HCO3 2.0e5 60:40/CH3CN: 5 mM NH4HCO3

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 297.2/159.2 amu from Sample.. Max. 1.3e4 cps. XIC of +MRM (14 pairs): 313.3/203.2 amu from Sample... Max. 5.2e5 cps.

Intensity, cps 9.55 Intensity, cps 12.12 1.3e4 10.62 5.2e5 4.0e5 1.0e4 Enilconazole 297.2/159.2 Praziquantel 313.3/203.2 18.03 60:40/CH CN: 5 mM NH HCO 90:10/MeOH: 5 mM NH4HCO3 5000.0 3 4 3 2.0e5 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 16 18 Time, min Time, min XIC of +MRM (1 pair): 384.1/337.9 amu from Sample... Max. 2730.5 cps. XIC of +MRM (2 pairs): 389.2/315.1 amu from Sample... Max. 1.1e4 cps. Intensity, cps Intensity, cps 16.96 20.56 6.18 6.82 2730 1.00e4 2000 Felodipine 384.1/337.9 Nisoldipin 389.2/315.1 40:60/CH CN: 5 mM NH HCO 1000 3 4 3 5000.00 40:60/CH3CN: 5 mM NH4HCO3 7.38 0 0.00 5 10 15 20 25 30 0.36 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (1 pair): 302.3/284.0 amu from Sample... Max. 1.2e5 cps. XIC of +MRM (10 pairs): 370.4/252.1 amu from Sample... Max. 1.4e6 cps. Intensity, cps Intensity, cps 4.69 6.59 5.36 1.4e6 7.52 1.00e5 Isoxsuprine 302.3/284.0 1.0e6 Lansoprzole 370.4/425.1 50:50/CH CN: 5 mM NH HCO 80:20/MeOH: 5 mM NH4HCO3 5.00e4 3 4 3 5.0e5 0.00 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (10 pairs): 231.3/127.2 amu from Sample... Max. 3.2e5 cps. XIC of +MRM (10 pairs): 346.3/125.1 amu from Sample... Max. 2.9e5 cps. Intensity, cps Intensity, cps 5.53 13.24 3.2e5 6.00 2.9e5 14.64 Metomidate 231.3/127.2 2.0e5 Metofoline 346.3/125.1 80:20/MeOH: 5 mM NH4HCO3 80:20/MeOH: 5 mM NH4HCO3 1.0e5 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 8a. Enantioresolution in RP LC/MS/MS on Lux Amylose-2

XIC of +MRM (9 pairs): 254.3/181.4 amu from Sample... Max. 5.9e5 cps. XIC of +MRM (9 pairs): 241.3/196.3 amu from Sample... Max. 3.2e6 cps. Intensity, cps 3.80 Intensity, cps 3.06 5.9e5 4.57 3.2e6 3.71 4.0e5 Nefopam 254.3/181.4 Pheniramine 241.3/196.3 60:40/CH CN: 5 mM NH HCO 60:40/CH CN: 5 mM NH HCO 2.0e5 3 4 3 3 4 3 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 231.3/127.2 amu from Sample... Max. 6.3e6 cps. XIC of +MRM (9 pairs): 220.2/128.1 amu from Sample... Max. 2.6e5 cps. Intensity, cps Intensity, cps 3.61 4.25 2.58 2.6e5 3.33 5.0e6 Metomidate 231.3/127.2 2.0e5 Ornidazole 220.2/128.1 60:40/CH3CN: 5 mM NH4HCO3 1.0e5 60:40/CH3CN: 5 mM NH4HCO3 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 370.4/252.1 amu from Sample... Max. 1.1e6 cps. XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 5393.8 cps. Intensity, cps Intensity, cps 2.92 3.96 4.50 1.00e6 5394 3.46 Lansoprizole 370.4/252.1 4000 Aminoglutethimid 233.2/188.3 5.00e5 60:40/CH3CN: 5 mM NH4HCO3 2000 60:40/CH3CN: 5 mM NH4HCO3 0.00 0 2 4 6 8 10 12 14 22.51 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 1.1e5 cps. XIC of +MRM (9 pairs): 231.1/115.1 amu from Sample... Max. 4.8e5 cps. Intensity, cps Intensity, cps 6.78 4.86 5.24 1.00e5 4.0e5 Motofoline 346.3/12.1 8.37 Kavain 231.1/115.1 5.00e4 60:40/CH3CN: 5 mM NH4HCO3 2.0e5 60:40/CH3CN: 5 mM NH4HCO3 4.16 0.00 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 2.5e5 cps. XIC of +MRM (9 pairs): 297.2/159.2 amu from Sample... Max. 1.2e4 cps. Intensity, cps Intensity, cps 3.53 4.06 5.10 5.50 1.00e4 2.0e5 Nomifensine 239.3/44.1 Enilconazole 297.2/159.2 60:40/CH CN: 5 mM NH HCO 1.0e5 60:40/CH3CN: 5 mM NH4HCO3 5000.00 3 4 3 2.10 0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 8b. Enantioresolution in RP LC/MS/MS on Lux Amylose-2

XIC of +MRM (14 pairs): 276.2/167.3 amu from Sample... Max. 5.0e5 cps. XIC of +MRM (14 pairs): 319.3/274.2 amu from Sample... Max. 1.4e6 cps. Intensity, cps Intensity, cps 3.38 3.39 1.4e6 Brompheniramine 319.3/274.2 4.0e5 Chlorpheniramine 276.2/167.3 70:30/CH3CN: 5 mM NH4HCO3 1.0e6 4.89 4.91 70:30/CH3CN: 5 mM NH4HCO3 2.0e5 5.0e5 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (12 pairs): 285.2/200.2 amu from Sample... Max. 1.5e5 cps. XIC of +MRM (12 pairs): 362.3/91.2 amu from Sample... Max. 1.0e4 cps. Intensity, cps Intensity, cps 3.79 4.60 1.5e5 1.00e4 Butaclamol 362.3/91.2 5.57 70:30/CH3CN: 5 mM NH4HCO3 1.0e5 5.00 Etozolin 285.2/200.2 70:30/CH3CN: 5 mM NH4HCO3 5000.00

0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (14 pairs): 417.1/159.1 amu from Sample... Max. 7.8e4 cps. XIC of +MRM (12 pairs): 342.3/116.3 amu from Sample... Max. 4.3e4 cps. Intensity, cps Intensity, cps 7.91 4.59 7.8e4 8.64 Propafenone 342.3/116.3 4.0e4 80:20/CH CN: 5 mM NH HCO Miconazole 417.1/159.1 3 4 3 5.0e4 9.27 70:30/CH3CN: 5 mM NH4HCO3 2.0e4

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (12 pairs): 293.4/164.3 amu from Sample... Max. 8.3e4 cps. XIC of +MRM (12 pairs): 381.1/125.1 amu from Sample... Max. 1.2e5 cps. Intensity, cps Intensity, cps 3.21 4.33 Econazole 381.1/125.1 8.3e4 4.65 3.83 Ambucetamid 293.4/164.3 1.00e5 80:20/CH3CN: 5 mM NH4HCO3 5.0e4 80:20/CH CN: 5 mM NH HCO 3 4 3 5.00e4 0.0 0.00 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (12 pairs): 500.3/142.3 amu from Sample... Max. 1.2e4 cps. XIC of +MRM (12 pairs): 299.3/104.3 amu from Sample... Max. 2.7e6 cps. Intensity, cps Intensity, cps 5.63 3.96 Isoamarine 299.3/104.3 Halofantrine 500.3/142.3 2.7e6 1.00e4 90:10/CH3CN: 5 mM NH4HCO3 90:10/CH3CN: 5 mM NH4HCO3 2.0e6 9.35 5000.00 10.52 1.0e6 0.00 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min Figure 9. Enantioseparation of Ketamin and Metabolite on Lux Amylose-2

XIC of +MRM (3 pairs): 238.1/125.0 amu from Sample 2 (Ketamines-50 % CH3CN) of Ketamines-50 %CH3CN Max. 6.1e4 cps. 5.05 6.0e4 5.59 5.0e4 Intensity, cps 4.0e4 Ketamine 238.1/125.0 3.0e4 50:50/CH3CN: 5 mM NH4HCO3 2.0e4 1.0e4 0.0 1 2 3 4 5 6 7 8 Time, min XIC of +MRM (3 pairs): 224.0/125.0 amu from Sample 2 (Ketamines-50%CH3CN) of Ketamines-50%CH3CN.w... Max. 1.6e5 cps. 4.48 1.5e5 4.95

Intensity, cps Norketamine 224.0/125.0 1.0e5 50:50/CH3CN: 5 mM NH4HCO3

5.0e4

0.0 1 2 3 4 5 6 7 8 Time, min XIC of +MRM (3 pairs): 242.1/129.3 amu from Sample 2 (Ketamines-50%CH3CN) of Ketamines-50%CH3CN.w... Max. 1.6e5 cps. 4.99 1.5e5 5.51 Intensity, cps Ketamine -d4 242.1/129.3 1.0e5 50:50/CH3CN: 5 mM NH4HCO3

5.0e4

0.0 1 2 3 4 5 6 7 8 Time, min Figure 10. Enantioseparation of Benzodiazepines and ß-blockers

XIC of +MRM (1 pair): 249.3/116.1 amu from Sample... Max. 1.1e5 cps. XIC of +MRM (12 pairs): 381.3/186.3 amu from Sample ... Max. 6.6e5 cps. Intensity, cps Intensity, cps 3.54 Pindolol 249.3/116.1 5.37 5.95 Bopidolol 381.3/186.3 1.00e5 60:40/CH3CN: 5 mM NH4HCO3 6.6e5 90:10/MeOH: 5 mM NH4HCO3 on Lux Cellulose-1 5.0e5 on Lux Cellulose-1 5.00e4 5.47

0.00 0.0 2 4 6 8 10 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 4.2e4 cps. XIC of +MRM (14 pairs): 381.3/186.3 amu from Sample ... Max. 6.0e4 cps. Intensity, cps Intensity, cps 4.27 Lorazepam 321.4/275.1 3.97 Bopidolol 381.3/186.3 4.0e4 60:40/CH3CN: 5 mM NH4HCO3 5.0e4 60:40/CH3CN: 5 mM NH4HCO3 6.63 on Lux Cellulose-1 on Lux Amylose-2 2.0e4 4.86 5.06 5.31 0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 10 12 14 Time, min Time, min XIC of +MRM (13 pairs): 287.1/241.3 amu from Sample... Max. 2.2e4 cps. XIC of +MRM (2 pairs): 224.2/147.2 amu from Sample... Max. 2.3e5 cps. Intensity, cps Oxazepam 287.1/241.3 Intensity, cps 4.85 3.68 Toliprolol 224.2/147.2 60:40/CH3CN: 5 mM NH4HCO3 2.0e4 2.0e5 70:30/MeOH: 5 mM NH4HCO3 on Lux Cellulose-1 on Lux Cellulose-1 8.68 5.14 1.0e4 1.0e5

0.0 0.0 2 4 6 8 10 12 14 2 4 6 8 Time, min Time, min XIC of +MRM (13 pairs): 301.3/255.1 amu from Sample... Max. 1.2e5 cps. XIC of +MRM (2 pairs): 266.3/72.2 amu from Sample... Max. 4.8e5 cps. Intensity, cps Intensity, cps 4.83 4.76 5.88 Temazepam 301.3/255.1 Oxprenolol 266.3/72.2 5.79 1.00e5 4.0e5 60:40/CH3CN: 5 mM NH4HCO3 70:30/MeOH: 5 mM NH4HCO3 5.00e4 Lux Cellulose-1 2.0e5 on Lux Cellulose-1 0.00 0.0 2 4 6 8 10 12 14 2 4 6 8 Time, min Time, min XIC of +MRM (1 pair): 225.0/165.2 amu from Sample... Max. 3881.0 cps. XIC of +MRM (7 pairs): 337.3/116.0 amu from Sample... Max. 1.2e5 cps. Intensity, cps Intensity, cps 25.77 3.53 3881 29.12 4.08 Nifenolol 225.0/165.2 1.00e5 Acebutolol 337.3/116.0 45:55/MeOH: 5 mM NH4HCO3 + 0.025 % DEA 40:60/CH3CN: 5 mM NH4HCO3 on Lux Cellulose-2 5.00e4 on Lux Amylose-2 0 0.00 5 10 15 20 25 30 2 4 6 8 10 12 14 Time, min Time, min

Chiral LC/MS/ MS experiments Three different polysaccharide- based chiral stationary phases Results and Discussion

Chiral LC/MS/MS experiments Effect of temperature on chiral resolution

Three different polysaccharide-based chiral stationary Lowering column temperature had little effect on phases — Lux Cellulose-1 (Cellulose tris[3,5- resolution with Lux Cellulose-2 (cellulose tris(3-chloro- dimethylphenylcarbamate]), Lux Cellulose-2 4-methylphenylcarbamate); peaks got broader as (Cellulose tris[3-chloro-4-methylphenylcarbamate]), retention increased (Figure 3 and 4). and Lux Amylose-2 (Amylose tris[5-chloro-2- In general, decreasing temperature produces slower methylphenylcarbamate]); see Figure 1) were explored in the reversed phase elution mode for the separation of a mass transfer kinetics, resulting in increased retention variety of basic and neutral compounds of pharmaceutical and decreased column efficiency. Our results (data not interest, in mobile phases made of 5 mM ammonium presented) show that the effect of column temperature bicarbonate or acetate with acetonitrile or methanol, and on chiral resolution varies from case to case; it is with MS/MS detection. unpredictable and not significant on any of the CSPs in the temperature range (5 ºC - 35 ºC) studied. Effect of mobile phase additives Effect of organic modifier on chiral resolution NH HCO and CH COONH additives: The responses 4 3 3 4 Acetonitrile or methanol organic modifier was used of representative racemates in 5 mM NH4HCO3/CH3CN mobile phase were compared to the responses in 0.1 in chiral RP HPLC separations. Increasing the eluting strength of the mobile phase will decrease retention % HCOOH/CH3CN (analysis on C18 stationary phase; and resolution as shown for nifenolol in and Figure 2). The results show that MS/MS responses Figure 3 Tolperison in Figure 7. However, once enantiomers eluted are comparable or higher in 5 mM NH4HCO3/CH3CN later than 10 minutes with only partial resolution, baseline compared to analyte responses in 0.1 % HCOOH/CH3CN. This proves that ammonium bicarbonate based mobile separation can be rarely achieved by decreasing % phases are favorable for MS/MS detection. organic modifier in the mobile phase.

The enantioseparation on Lux CSPs was evaluated in In our study, acetonitrile provides more successful chiral resolution than methanol on Lux CSP in RP mode. both 5 mM CH3COONH4/CH3CN and 5 mM NH4HCO3/ CH3CN (Figure 5). In general, NH4HCO3 provided similar Chiral LC/MS/MS applications or occasionally superior resolution to CH3COONH4 as mobile phase additive. Figures 5-10 demonstrate more than 50 chiral separations on Lux CSPs. Most compounds evaluated here eluted in Diethylamine (DEA) additive: DEA is a commonly used less than 10 min with baseline resolution in mobile phases additive in chiral HPLC with polysaccharide derivatives; of various eluting strength. The results show that Lux unfortunately, it severely suppresses analyte responses in 3 µm Cellulose-1 was most successful in separating ESI+ MS/MS even at low concentration levels as 0.025 % benzodiazepines and ß-blockers, Lux 3 µm (Figures 3-4). Addition of DEA into mobile phase could Cellulose-2 in separating imidazoles, and Lux improve enantioresolution for very basic compounds (e.g. 3 µm Amylose-2 in separating antihistamines and ß-blockers and tricyclic antidepressants); however DEA imidazoles. does not affect the enantioresolution of benzodiazepines, imidazoles or neutral stereoisomers (Figure 6-9). For all these compunds, baseline separation can be achieved without DEA. Conclusions

• More than 50 chiral LC/MS/MS analyses are demonstrated on the polysaccharide- based CSPs stationary phases Lux 3 µm Cellulose-1, Lux 3 µm Cellulose-2, and Lux 3 µm Amylose-2 in the reversed phase elution mode.

• Ammonium bicarbonate is the preferred buffer salt with ESI+ MS/MS detection for most basic pharmaceutical stereoisomers. Ammonium acetate is a viable alternative to ammonium bicarbonate but it is less successful in providing baseline resolution.

• Diethylamine (as additional additive) can improve the chiral resolution of strong basic compounds but it has a negative effect on analyte response in ESI+ MS/MS even at low concentration levels (e.g. 0.025 %).

• Decreasing column temperature may not improve chiral resolution on partially separated strong basic stereoisomers because of peak broadening with delayed retention.

• Increasing the organic modifier (CH3CN or MeOH) in the RP mobile phase has the expected effect: it decreases retention and enantioselectivity; adjusting % organic modifier is essential to optimizing chiral resolution.

Trademarks Lux and Gemini-NX are registered trademarks of Phenomenex, Inc in the United States, European Union, and other jurisdictions. TurbolonSpary is a registered trademark of Applied Biosystems. Gemini-NX is patented by Phenomenex U.S. Patent No. 7,563,367