PO
878
2061
1_L
_2
Chiral LC/MS/MS Analysis with Polysaccharide-based Stationary Phases for Basic and Neutral Stereoisomeric Pharmaceutical Compounds
Liming Peng, Tivadar Farkas and Swapana Jayapaian
Phenomenex, Inc., 411 Madrid Ave.,Torrance, CA 90501 USA (www.phenomenex.com)
The hyphenation of the resolving power of chiral HPLC with the sensitivity of MS detection is highly desired in drug metabolism and pharmacokinetic studies of stereoisomers in the drug discovery process. Derivatives of polysaccharides are the most widely used chiral stationary phases due to their wide chiral recognition ability, high loading capacity, and durability. As normal phase is favorable for the principal mechanism of chiral recognition – hydrogen bonding interaction – the majority of chiral separations with polysaccharide phases are performed in normal phase using hexane and alcohol modifiers as mobile phase components. However these mobile phases are highly flammable and are not
compatible with atmospheric pressure ionization (API) MS ion sources. Developing rapid chiral LC separations compatible with MS ionization interfaces while preserving both chromatographic resolution and sensitivity in MS detection has proven to be a great challenge to analytical scientists.
We present the results of a systematic feasibility study of using polysaccharide-based chiral stationary phases (CSPs) coupled with API-MS/MS detection for the analysis of various pharmaceutical racemates in reversed phase (RP) elution mode.
Introduction
Experimental Conditions
HPLC System: HP 1100 series (www.agilent.com)
Pump: G1312A (Binary Pump)
Autosampler: G1329A ALS
MS Detector: API 3000 LC/MS/MS with ESI (TurboIonSpray®) (www. Appliedbiosystems.com)
TurbolonSpray - ESI, Positive Ion Mode; MRM; heater gas flow 5000 cc/min; heater temperature 400 ˚C.
Flow Rate: 0.2 mL/minInjection Volume: 5 µL
Concentration: 0.2 – 1.0 µg/mL of racematesColumns: Lux 3 µm Cellulose-1
150 x 2.0 mmLux 3 µm Cellulose-2 150 x 2.0 mmLux 3 µm Amylose -2 150 x 2.0 mm Gemini®-NX 3 µm C1850 x 2.0 mm
Mobile Phases: 1. Acetonitrile/Methanol : 5 mM NH4HCO3
2. Acetonitrile/Methanol: 5 mM NH4HCO3 + 0.025 % DEA*
3. Acetonitrile/Methanol: 5 mM CH3COONH4
* DEA - Diethylamine
Figure 2. LC/MS/MS Responses of Representative Racemates
Ketam
ine
Isoxs
uprin
e
Nisoldipine
Trim
ipram
ine
Norke
tam
ine
Nifeno
lol
0.E+00
5.E+06
1.E+07
2.E+07
2.E+07
3.E+07
3.E+07
4.E+07
4.E+07
LC/M
S/M
S r
esp
ons
es0.1 % Formic acid/Acetonitrile5 mM Ammonium Bircarbonate/Acetonitrile
Ketam
ine
Isoxs
uprin
e
Nisoldipine
Trim
ipram
ine
Norke
tam
ine
Nifeno
lol
0.E+00
1.E+06
2.E+06
3.E+06
4.E+06
5.E+06
6.E+06
7.E+06
8.E+06
9.E+06
1.E+07
LC/M
S/M
S r
esp
ons
es
0.1 % Formic acid/CH3CN
5 mM NH4HCO3/CH3CN
Conc.: 200 ng/mL Column: Gemini-NX 3 µm50 x 2.0 mm
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.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
Intensity, cps
8.838.42
Nifenolol 225.0/165.2Lux Cellulose-260:40/MeOH: 5 mM NH4HCO3
XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 1.8e5 cps.
2 4 6 8 10 12 14 16 18 20 22 24Time, min
0.0
5.0e4
1.0e5
1.5e5
1.8e5Intensity, cps
15.18 16.09
Trimipramine 295.4/100.0Lux Cellulose-250:50/CH3CN: 5 mM NH4HCO3
XIC of +MRM (1 pair): 225.0/165.2 amu from Sample ... Max. 1.6e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5000.0
1.0e4
1.5e4
Intensity, cps
8.15 8.77
Nifenolol 225.0/165.2Lux Cellulose-2 60:40//MeOH: 5 mM NH4HCO3 +0.025 % DEA
XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 1.5e5 cps.
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26Time, min
0.0
5.0e4
1.0e5
1.5e5
Intensity, cps
22.71 24.21
24.44Trimipramine 295.4/100.0Lux Cellulose-2 45:55/CH3CN: 5 mM NH4HCO3
XIC of +MRM (1 pair): 225.0/165.2 amu from Sample... Max. 3881.0 cps.
5 10 15 20 25 30Time, min
0
1000
2000
3000
3881
Intensity, cps
25.77
29.12Nifenolol 225.0/165.2Lux Cellulose-2 45:55/MeOH: 5 mM NH4HCO3 + 0.025 % DEA35 min
XIC of +MRM (1 pair): 295.4/100.0 amu from Sample ... Max. 4.8e4 cps.
2 4 6 8 10 12 14 16 18 20 22 24Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
4.8e4
Intensity, cps
18.69 19.92
Trimipramine 295.4/100.0Lux Cellulose-250:50/CH3CN: 5 mM NH4HCO3 +0.025 % DEA
Broader peaks & delayed RT
Decreased signal
Decreased signal
XIC of +MRM (4 pairs): 180.0/124.1 amu from Sample... Max. 4.4e5 cps.
2 4 6 8 10Time, min0.0
1.0e5
2.0e5
3.0e5
4.0e54.4e5
Intensity, cps
4.66
5.52
Adrenaline 180.0/124.1Lux Cellulose-250:50/MeOH: 5 mM NH4HCO3
@ ambient
XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 8.8e4 cps.
2 4 6 8Time, min0.0
2.0e4
4.0e4
6.0e4
8.0e48.8e4
Intensity, cps
5.836.52
Lorazepam 321.4/275.1Lux Cellulose-280:20/MeOH: 5 mM NH4HCO3
@ ambient
XIC of +MRM (4 pairs): 180.0/124.1 amu from Samplee... Max. 1.9e5 cps.
2 4 6 8 10Time, min0.0
5.0e4
1.0e5
1.5e5
1.9e5
Intensity, cps
6.61
8.50
2.95
Adrenaline 180.0/124.1Lux Cellulose-250:50/MeOH: 5 mM NH4HCO3
@ 5ºC
XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 1.9e4 cps.
2 4 6 8 10Time, min
0.0
5000.0
1.0e4
1.5e4
1.9e4
Intensity, cps
7.819.03
Lorazepam 321.4/275.1Lux Cellulose-280:20/MeOH: 5 mM NH4HCO3
@ 5ºC
XIC of +MRM (4 pairs): 180.0/124.1 amu from Sample... Max. 2.0e5 cps.
2 4 6 8 10Time, min0.0
5.0e4
1.0e5
1.5e5
2.0e5
Intensity, cps
4.55
5.40
Adrenaline 180.0/124.1Lux Cellulose-250:50/MeOH: 5 mM NH4HCO3 + 0.025 % DEA@ ambient
XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 2313.2 cps.
2 4 6 8 10Time, min
0
500
1000
1500
2000
2313
Intensity, cps
5.666.32
Lorazepam 321.4/275.1Lux Cellulose-280:20/MeOH: 5 mM NH4HCO3 +0.025 % DEA@ ambient
Figure 4. Effects of Temperature and Additive on Enantiorecognition
Decreased signal
Broad peaks & delayed retention
XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 1.6e4 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
1.6e4
Intensity, cps
4.303.75
2.74
Aminoglutethimide 233.2/188.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 4120.0 cps.
2 4 6 8 10 12 14Time, min
0
2000
4000
Intensity, cps
3.453.96
Aminoglutethimide 233.2/188.360:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 181.2/141.2 amu from Sampl... Max. 3.3e4 cps.
2 4 6 8 10 12 14Time, min
0.0
3.3e4
Intensity, cps
4.415.94
Orphenadrin 181.2/141.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 181.2/141.2 amu from Sample... Max. 4.1e4 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e4
4.0e4
Intensity, cps
2.632.43
Orphenadrin 181.2/141.260:40/CH3CN: 5 mM NH4AC
XIC of +MRM (13 pairs): 366.1/132.2 amu from Sample... Max. 2.9e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
2.9e5
Intensity, cps
4.76
6.17Indapamide 366.1/132.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 366.1/132.2 amu from Sample... Max. 2.3e4 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
2.0e4
Intensity, cps
4.33
5.65 Indapamide 366.1/132.260:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 2.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e52.6e5
Intensity, cps
8.73 9.41
Motofoline 346.3/125.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 1.4e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e4
1.0e5
1.4e5
Intensity, cps
4.894.63
Motofoline 346.3/125.160:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 370.4/252.1 amu from Sample... Max. 2.1e6 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e6
2.0e6
Intensity, cps
3.86
Lansoprizole 370.4/252.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 370.4/252.1 amu from Sampl... Max. 2.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e52.6e5
Intensity, cps
3.44
Lansoprizole 370.4/252.160:40/CH3CN: 5 mM NH4AC
Figure 5a. 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.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
2.0e42.5e4
Intensity, cps
3.97 4.58
5.84 6.34
Zopiclone 245.1/217.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 245.1/217.3 amu from Sample... Max. 8660.0 cps.
2 4 6 8 10 12 14Time, min
0
5000
8660
Intensity, cps
3.52 4.06
5.885.41Zopiclone 245.1/217.360:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 314.3/176.2 amu from Sample... Max. 4.1e6 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e6
4.0e6
Intensity, cps
4.406.56
Reboxetine 314.3/176.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 314.3/176.2 amu from Sample... Max. 3.1e6 cps.
2 4 6 8 10 12 14Time, min
0.0
3.1e6
Intensity, cps
3.862.92
Reboxetine 314.3/176.260:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 3.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
3.6e5
Intensity, cps
5.41 5.93Nomifensine 239.3/44.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 2.4e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
Intensity, cps
4.17 4.56
Nomifensine 239.3/44.160:40/CH3CN: 5 mM NH4AC
XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 5.0e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
6.98 7.96
Mebeverine 430.4/149.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 6.7e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e56.7e5
Intensity, cps
2.77
Mebeverine 430.4/149.160:40/CH3CN: 5 mM NH4AC
XIC of +MRM (14 pairs): 272.4/129.4 amu from Samp... Max. 5.1e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e55.1e5
Intensity, cps
6.74 7.02
Napropamide 272.4/129.460:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 272.4/129.4 amu from Samp... Max. 2.3e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
Intensity, cps
6.456.18
Napropamide 272.4/129.460:40/CH3CN: 5 mM NH4AC
Figure 5b. Effect of Buffer on Chiral Resolution in RP LC/MS/MS on Lux Cellulose-1
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.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
1.0e6
1.5e6
Intensity, cps
4.01
5.63 Dimethindene 293.4/248.3in 90:10/MeOH: 5 mM NH4HCO3
XIC of -MRM (4 pairs): 420.0/289.1 amu from Sample... Max. 1.1e5 cps.
2 4 6 8Time, min
0.00
5.00e4
1.00e5
Intensity, cps
3.51 3.93Bendroflumethiazde 420.0/289.1(negative mode)in 80:20/MeOH: 5 mM NH4HCO3
XIC of +MRM (11 pairs): 285.2/200.2 amu from Sample... Max. 9.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
9.5e5
Intensity, cps
6.497.87Etozolin 285.2/200.2
in 90:10/MeOH: 5 mM NH4HCO3
XIC of +MRM (3 pairs): 276.3/124.2 amu from Sample... Max. 8.3e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
8.3e5
Intensity, cps
3.33 4.09
Homatropine 276.3/124.2in 80:20/MeOH: 5 mM NH4HCO3
XIC of +MRM (11 pairs): 299.4/104.2 amu from Sample... Max. 1.5e6 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e6
1.5e6
Intensity, cps
4.437.01
Isoamarine 299.4/104.2in 90:10/MeOH: 5 mM NH4HCO3
XIC of +MRM (6 pairs): 272.3/44.1 amu from Sample ... Max. 4.4e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
6.82 8.39
Nisoxetine 272.3/44.1in 40:60/CH3CN: 5 mM NH4HCO3
XIC of +MRM (11 pairs): 265.3/208.3 amu from Sample... Max. 1.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
1.6e5
Intensity, cps
6.608.13
Mianserin 265.3/208.3in 90:10/MeOH: 5 mM NH4HCO3
XIC of +MRM (6 pairs): 264.3/58.2 amu from Sample ... Max. 1.0e6 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e5
1.00e6
Intensity, cps
6.135.60
Tramadol 264.3/58.2in 40:60/CH3CN: 5 mM NH4HCO3
XIC of +MRM (11 pairs): 500.3/142.3 amu from Sample... Max. 1.6e4 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
1.6e4
Intensity, cps
10.67 12.44
Halofantrine 500.3/142.3in 90:10/MeOH: 5 mM NH4HCO3
XIC of +MRM (3 pairs): 391.2/201.1 amu from Sample... Max. 4.1e6 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e6
4.0e6
Intensity, cps
7.98 8.59
Meclizine 391.2/201.1in 70:30/CH3CN: 5 mM NH4HCO3
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.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
Intensity, cps
5.55 6.54
Tolperisone 246.3/98.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 299.4/104.2 amu from Sample... Max. 2.8e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e52.8e5
Intensity, cps
5.16 5.57
Isoamarine 299.4/104.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 251.3/132.3 amu from Sample... Max. 9.1e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e4
9.1e4
Intensity, cps
4.77
Methaqualone 251.3/132.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 272.4/129.4 amu from Sample... Max. 4.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
6.63 7.15
Napropamide 272.4/129.460:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 259.1/84.2 amu from Sample... Max. 1.4e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5000.0
1.0e41.4e4
Intensity, cps
3.78
2.62
Thalidomide 259.1/84.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 328.3/120.2 amu from Sample... Max. 3021.8 cps.
2 4 6 8 10 12 14Time, min
0
3022
Intensity, cps
2.80
5.80Omeprazole 328.3/120.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 205.1/178.3 amu from Sample... Max. 3.8e5 cps.
2 4 6 8 10 12 14Time, min
0.0
3.8e5
Intensity, cps
4.885.44
Tetramisole 205.1/178.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 398.4/124.2 amu from Sample... Max. 1.0e6 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e5
1.00e6
Intensity, cps
6.447.29
Diperodon 398.4/124.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 430.4/149.1 amu from Sample... Max. 5.4e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e55.4e5
Intensity, cps
6.85 7.34
9.05
Mebeverine 430.4/149.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 2.0e4 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
2.0e4
Intensity, cps
3.61 3.96
3.24 4.30 13.78
Aminoglutethimide 233.2/188.360:40/CH3CN: 5 mM NH4HCO3
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.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e55.5e5
Intensity, cps
3.983.63
Tolperisone 246.3/98.280:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 299.3/104.3 amu from Sample... Max. 3.4e5 cps.
2 4 6 8 10 12 14Time, min
0.0
3.4e5
Intensity, cps
3.12
Isoamarine 299.3/104.380:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 311.2/243.3 amu from Sample... Max. 4.1e6 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e6
4.0e6
Intensity, cps
5.42 6.08
Bifonazole 311.2/243.380:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 285.2/200.2 amu from Sample... Max. 7.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
7.5e5
Intensity, cps
4.155.69
Etozolin 285.2/200.280:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 362.3/91.2 amu from Sample... Max. 5.1e4 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e4
4.0e45.1e4
Intensity, cps
4.566.71
Butaclamol 362.3/91.280:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 466.2/184.0 amu from Sample... Max. 7.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
7.5e5
Intensity, cps
4.265.19
Cisapride 466.2/184.080:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 293.4/164.3 amu from Sample... Max. 3.7e5 cps.
2 4 6 8 10 12 14Time, min
0.0
3.7e5
Intensity, cps
5.39
7.20
Ambucetamid 293.4/164.380:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 381.1/125.1 amu from Sample... Max. 7.3e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
7.3e5
Intensity, cps
7.398.67
Econazole 381.1/125.180:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 417.1/159.1 amu from Sample... Max. 1.9e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
1.9e5
Intensity, cps
10.2012.07
Miconaxole 417.1/159.180:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (13 pairs): 500.3/142.3 amu from Sample... Max. 1.0e4 cps.
2 4 6 8 10 12 14Time, min
0.00
5000.00
1.00e4
Intensity, cps
11.5910.95
12.48
Halofantrine 500.3/142.380:20/CH3CN: 5 mM NH4HCO3
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.
2 4 6 8 10 12 14Time, min
0.0
2.0e6
4.0e6
Intensity, cps
4.06 4.58
Reboxetine 314.3/176.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample... Max. 4.1e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
4.17 5.12
Nomifensine 239.3/44.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 297.2/159.2 amu from Sample.. Max. 1.3e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5000.0
1.0e41.3e4
Intensity, cps
9.55 10.62
Enilconazole 297.2/159.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 313.3/203.2 amu from Sample... Max. 5.2e5 cps.
2 4 6 8 10 12 14 16 18Time, min
0.0
2.0e5
4.0e55.2e5
Intensity, cps
12.12
18.03Praziquantel 313.3/203.290:10/MeOH: 5 mM NH4HCO3
XIC of +MRM (1 pair): 384.1/337.9 amu from Sample... Max. 2730.5 cps.
5 10 15 20 25 30Time, min
0
1000
20002730
Intensity, cps
16.96 20.56
Felodipine 384.1/337.940:60/CH3CN: 5 mM NH4HCO3
XIC of +MRM (2 pairs): 389.2/315.1 amu from Sample... Max. 1.1e4 cps.
2 4 6 8 10 12 14Time, min
0.00
5000.00
1.00e4
Intensity, cps
6.18 6.82
7.38
0.36
Nisoldipin 389.2/315.140:60/CH3CN: 5 mM NH4HCO3
XIC of +MRM (1 pair): 302.3/284.0 amu from Sample... Max. 1.2e5 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e4
1.00e5
Intensity, cps
4.69 5.36
Isoxsuprine 302.3/284.050:50/CH3CN: 5 mM NH4HCO3
XIC of +MRM (10 pairs): 370.4/252.1 amu from Sample... Max. 1.4e6 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
1.0e61.4e6
Intensity, cps
6.59 7.52
Lansoprzole 370.4/425.180:20/MeOH: 5 mM NH4HCO3
XIC of +MRM (10 pairs): 231.3/127.2 amu from Sample... Max. 3.2e5 cps.
2 4 6 8 10 12 14Time, min
0.0
3.2e5
Intensity, cps
5.53 6.00
Metomidate 231.3/127.280:20/MeOH: 5 mM NH4HCO3
XIC of +MRM (10 pairs): 346.3/125.1 amu from Sample... Max. 2.9e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
2.9e5
Intensity, cps
13.2414.64
Metofoline 346.3/125.180:20/MeOH: 5 mM NH4HCO3
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.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
5.9e5
Intensity, cps
3.804.57
Nefopam 254.3/181.460:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 241.3/196.3 amu from Sample... Max. 3.2e6 cps.
2 4 6 8 10 12 14Time, min
0.0
3.2e6
Intensity, cps
3.063.71
Pheniramine 241.3/196.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 231.3/127.2 amu from Sample... Max. 6.3e6 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e6
Intensity, cps
3.61 4.25
Metomidate 231.3/127.260:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 220.2/128.1 amu from Sample... Max. 2.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e52.6e5
Intensity, cps
2.583.33
Ornidazole 220.2/128.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 370.4/252.1 amu from Sample... Max. 1.1e6 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e5
1.00e6
Intensity, cps
2.92
3.46 Lansoprizole 370.4/252.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 233.2/188.3 amu from Sample... Max. 5393.8 cps.
2 4 6 8 10 12 14Time, min
0
2000
40005394
Intensity, cps
3.96 4.50
2.51
Aminoglutethimid 233.2/188.360:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 346.3/125.1 amu from Sample... Max. 1.1e5 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e4
1.00e5
Intensity, cps
6.78
8.37Motofoline 346.3/12.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 231.1/115.1 amu from Sample... Max. 4.8e5 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
4.86 5.24
4.16
Kavain 231.1/115.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 239.3/44.1 amu from Sample ... Max. 2.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
2.0e5
Intensity, cps
4.063.53
2.10
Nomifensine 239.3/44.160:40/CH3CN: 5 mM NH4HCO3
XIC of +MRM (9 pairs): 297.2/159.2 amu from Sample... Max. 1.2e4 cps.
2 4 6 8 10 12 14Time, min
0.00
5000.00
1.00e4
Intensity, cps
5.10 5.50
Enilconazole 297.2/159.260:40/CH3CN: 5 mM NH4HCO3
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.
2 4 6 8 10 12 14Time, min
0.0
2.0e5
4.0e5
Intensity, cps
3.39
4.91Chlorpheniramine 276.2/167.370:30/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 319.3/274.2 amu from Sample... Max. 1.4e6 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e5
1.0e61.4e6
Intensity, cps
3.38
4.89
Brompheniramine 319.3/274.270:30/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 285.2/200.2 amu from Sample... Max. 1.5e5 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e5
1.5e5
Intensity, cps
3.79
5.00 Etozolin 285.2/200.270:30/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 362.3/91.2 amu from Sample... Max. 1.0e4 cps.
2 4 6 8 10 12 14Time, min
0.00
5000.00
1.00e4
Intensity, cps
4.605.57
Butaclamol 362.3/91.270:30/CH3CN: 5 mM NH4HCO3
XIC of +MRM (14 pairs): 417.1/159.1 amu from Sample... Max. 7.8e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e4
7.8e4
Intensity, cps
7.918.64
Miconazole 417.1/159.170:30/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 342.3/116.3 amu from Sample... Max. 4.3e4 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e4
4.0e4
Intensity, cps
4.59
9.27
Propafenone 342.3/116.380:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 293.4/164.3 amu from Sample... Max. 8.3e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e4
8.3e4
Intensity, cps
3.21
3.83 Ambucetamid 293.4/164.380:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 381.1/125.1 amu from Sample... Max. 1.2e5 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e4
1.00e5
Intensity, cps
4.334.65
Econazole 381.1/125.180:20/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 500.3/142.3 amu from Sample... Max. 1.2e4 cps.
2 4 6 8 10 12 14Time, min
0.00
5000.00
1.00e4
Intensity, cps
5.63
10.52
Halofantrine 500.3/142.390:10/CH3CN: 5 mM NH4HCO3
XIC of +MRM (12 pairs): 299.3/104.3 amu from Sample... Max. 2.7e6 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e6
2.0e62.7e6
Intensity, cps
3.96
9.35
Isoamarine 299.3/104.390:10/CH3CN: 5 mM NH4HCO3
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.
1 2 3 4 5 6 7 8Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
Intensity, cps
5.055.59
Ketamine 238.1/125.050:50/CH3CN: 5 mM NH4HCO3
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.
1 2 3 4 5 6 7 8Time, min
0.0
5.0e4
1.0e5
1.5e5
Intensity, cps
4.48
4.95
Norketamine 224.0/125.050:50/CH3CN: 5 mM NH4HCO3
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.
1 2 3 4 5 6 7 8Time, min
0.0
5.0e4
1.0e5
1.5e5
Intensity, cps
4.995.51
Ketamine -d4 242.1/129.350:50/CH3CN: 5 mM NH4HCO3
Figure 10. Enantioseparation of Benzodiazepines and ß-blockers
XIC of +MRM (1 pair): 249.3/116.1 amu from Sample... Max. 1.1e5 cps.
2 4 6 8 10Time, min
0.00
5.00e4
1.00e5
Intensity, cps
3.54
5.47
Pindolol 249.3/116.160:40/CH3CN: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (12 pairs): 381.3/186.3 amu from Sample ... Max. 6.6e5 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e56.6e5
Intensity, cps
5.955.37 Bopidolol 381.3/186.390:10/MeOH: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (2 pairs): 321.4/275.1 amu from Sample... Max. 4.2e4 cps.
2 4 6 8 10 12 14Time, min
0.0
2.0e4
4.0e4
Intensity, cps
4.27
6.63
5.06 5.31
Lorazepam 321.4/275.160:40/CH3CN: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (14 pairs): 381.3/186.3 amu from Sample ... Max. 6.0e4 cps.
2 4 6 8 10 12 14Time, min
0.0
5.0e4
Intensity, cps
3.97
4.86
Bopidolol 381.3/186.360:40/CH3CN: 5 mM NH4HCO3on Lux Amylose-2
XIC of +MRM (13 pairs): 287.1/241.3 amu from Sample... Max. 2.2e4 cps.
2 4 6 8 10 12 14Time, min
0.0
1.0e4
2.0e4
Intensity, cps
4.85
8.68
Oxazepam 287.1/241.360:40/CH3CN: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (2 pairs): 224.2/147.2 amu from Sample... Max. 2.3e5 cps.
2 4 6 8Time, min
0.0
1.0e5
2.0e5
Intensity, cps
3.68
5.14
Toliprolol 224.2/147.270:30/MeOH: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (13 pairs): 301.3/255.1 amu from Sample... Max. 1.2e5 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e4
1.00e5
Intensity, cps
4.83 5.88 Temazepam 301.3/255.160:40/CH3CN: 5 mM NH4HCO3Lux Cellulose-1
XIC of +MRM (2 pairs): 266.3/72.2 amu from Sample... Max. 4.8e5 cps.
2 4 6 8Time, min
0.0
2.0e5
4.0e5
Intensity, cps
4.765.79Oxprenolol 266.3/72.2
70:30/MeOH: 5 mM NH4HCO3on Lux Cellulose-1
XIC of +MRM (1 pair): 225.0/165.2 amu from Sample... Max. 3881.0 cps.
5 10 15 20 25 30Time, min
0
3881
Intensity, cps
25.7729.12
Nifenolol 225.0/165.245:55/MeOH: 5 mM NH4HCO3 + 0.025 % DEAon Lux Cellulose-2
XIC of +MRM (7 pairs): 337.3/116.0 amu from Sample... Max. 1.2e5 cps.
2 4 6 8 10 12 14Time, min
0.00
5.00e4
1.00e5
Intensity, cps
3.534.08
Acebutolol 337.3/116.040:60/CH3CN: 5 mM NH4HCO3on Lux Amylose-2
Chiral LC/MS/M S e x p e r i -ments
T h re e d i f f e re n t polysaccharide-b a s e d c h i r a l stationary phases
Chiral LC/MS/MS experiments
Three different polysaccharide-based chiral stationary phases — Lux Cellulose-1 (Cellulose tris[3,5-dimethylphenylcarbamate] ), Lux Cel lu lose-2 (Cellulose tris[3-chloro-4-methylphenylcarbamate]), and Lux Amylose-2 (Amylose tris[5-chloro-2-methylphenylcarbamate]); see Figure 1) were explored in the reversed phase elution mode for the separation of a variety of basic and neutral compounds of pharmaceutical interest, in mobile phases made of 5 mM ammonium bicarbonate or acetate with acetonitrile or methanol, and with MS/MS detection.
Effect of mobile phase additives
NH4HCO3 and CH3COONH4 additives: The responses of representative racemates in 5 mM NH4HCO3/CH3CN mobile phase were compared to the responses in 0.1 % HCOOH/CH3CN (analysis on C18 stationary phase; Figure 2). The results show that MS/MS responses are comparable or higher in 5 mM NH4HCO3/CH3CN compared to analyte responses in 0.1 % HCOOH/CH3CN. This proves that ammonium bicarbonate based mobile phases are favorable for MS/MS detection.
The enantioseparation on Lux CSPs was evaluated in both 5 mM CH3COONH4/CH3CN and 5 mM NH4HCO3/CH3CN (Figure 5). In general, NH4HCO3 provided similar or occasionally superior resolution to CH3COONH4 as mobile phase additive.
Diethylamine (DEA) additive: DEA is a commonly used additive in chiral HPLC with polysaccharide derivatives; unfortunately, it severely suppresses analyte responses in ESI+ MS/MS even at low concentration levels as 0.025 % (Figures 3-4). Addition of DEA into mobile phase could improve enantioresolution for very basic compounds (e.g. ß-blockers and tricyclic antidepressants); however DEA 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.
Effect of temperature on chiral resolution
Lowering column temperature had little effect on resolution with Lux Cellulose-2 (cellulose tris(3-chloro-4-methylphenylcarbamate); peaks got broader as retention increased (Figure 3 and 4).
In general, decreasing temperature produces slower mass transfer kinetics, resulting in increased retention and decreased column efficiency. Our results (data not presented) show that the effect of column temperature on chiral resolution varies from case to case; it is unpredictable and not significant on any of the CSPs in the temperature range (5 ºC - 35 ºC) studied.
Effect of organic modifier on chiral resolution
Acetonitrile or methanol organic modifier was used in chiral RP HPLC separations. Increasing the eluting strength of the mobile phase will decrease retention and resolution as shown for nifenolol in Figure 3 and Tolperison in Figure 7. However, once enantiomers eluted later than 10 minutes with only partial resolution, baseline separation can be rarely achieved by decreasing % organic modifier in the mobile phase.
In our study, acetonitrile provides more successful chiral resolution than methanol on Lux CSP in RP mode.
Chiral LC/MS/MS applications
Figures 5-10 demonstrate more than 50 chiral separations on Lux CSPs. Most compounds evaluated here eluted in less than 10 min with baseline resolution in mobile phases of various eluting strength. The results show that Lux 3 µm Cellulose-1 was most successful in separating benzodiazepines and ß-blockers, Lux 3 µm Cellulose-2 in separating imidazoles, and Lux 3 µm Amylose-2 in separating antihistamines and imidazoles.
Results and Discussion
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 (CH• 3CN 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.
TrademarksLux and Gemini-NX are registered trademarks of Phenomenex, Inc in the United States, European Union, and other jurisdictions. TurbolonSpary is a regis-tered trademark of Applied Biosystems. Gemini-NX is patented by Phenomenex U.S. Patent No. 7,563,367
© 2011 Phenomenex, Inc. All rights reserved.
