TN-0103 APPLICATIONS · TN-0103 For additional technical notes, visit Page 1 of 8 APPLICATIONS A...
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TN-0103 For additional technical notes, visit www.phenomenex.com Page 1 of 8 APPLICATIONS A Sensitive Extraction Method for Nicotine and Metabolites from Urine using Simplified Liquid Extraction (SLE) Xianrong (Jenny) Wei, Ann Montross, and Sean Orlowicz Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA SLE Conditions Load: Load pretreated sample onto the Novum MAX 96-Well Plate (Phenomenex Part No.: 8E-S138-5GA) and apply 5” Hg of vacuum to initiate loading. Stop vacuum and wait 5 minutes. Elute: Dispense 600 µL of 1 % Formic acid in DCM/IPA (95:5). Re- peat two more times. Collect eluents in a collection plate (2 mL). Dry Down: Evaporate the final extract to complete dryness under a slow stream of N 2 at 45 °C. Reconstitute: Add 300 µL of 20 mM Ammonium Bicarbonate, pH 8.2/Methanol (90:10) and vortex. This method has been validated and shown acceptable accuracy and precision using a small volume of human urine in the analy- sis of nicotine and its metabolites (anabasine, nornicotine, cotinine and 3-hydroxycotinine). The modified method employed uses a Novum ™ SLE MAX 96-well plate with pH adjusted organic elution solvent, and a Kinetex ® EVO C18 column with high pH mobile phase to increase analyte recoveries and sensitivity using LC/MS/MS. Introduction Analysis of nicotine in urine has become more widespread with the increased use of tobacco-less products, such as nicotine lozenges, vaporizers, and gums. Analyzing nicotine metabolites (anabasine, nornicotine, cotinine and 3-hydroxycotinine) allows researchers to distinguish between an active smoker and some- one using tobacco-less products. The presence of tobacco me- tabolites, anabasine and nornicotine, in urine is an indication of an active smoker. The goals of this study were to simplify the extraction process using a Simplified Liquid Extraction (SLE) method, increase re- coveries, and demonstrate the assay accuracy, precision and lin- earity for all compounds under (GLP) Good Laboratory Pratice guidance. The endogenous level of nicotine and its metabolites were monitored during the method development for the deter- mination of LLOQ quantification level purpose. The method uses higher pH mobile phase at pH 8.2 to ensure the separations of all compounds, and therefore the Kinetex EVO C18 column was chosen for its widely stable pH range and chromatography. Experimental Conditions Reagents and Chemicals All solvents and reagents were HPLC or analytical grade. HPLC Grade methanol was purchased from Honeywell Burdick & Jackson ® (Muskegon, MI), Milli-Q ® water was used for sample preparation. HPLC Grade water was purchased from Honeywell Burdick & Jackson and used to prepare the LC mobile phase. Reference standards were purchased from Cerilliant ® Corporation. Agilent ® 1260 pumps and autosampler were used along with a SCIEX QTRAP ® 4500, positive polarity, ESI for detection. 200 µL of 2 % NH 4 OH in water and 50 µL of mixed deuterated in- ternal standards (200 ng/mL each in water) were added to 100 µL of human urine (spiked with analytes and/or blank) and mixed. LC/MS/MS Conditions: Column: Kinetex ® 2.6 µm EVO C18 Dimensions: 100 x 3.0 mm Part No.: 00D-4725-Y0 Mobile Phase: A: 20 mM Ammonium Bicarbonate, pH 8.2 B: Methanol Gradient: Time (min) B (%) 0 10 3 90 5 90 5.01 10 6. 10 Flow Rate: 0.75 mL/min Temperature: Ambient Detection: SCIEX QTRAP 4500 Turbo ™ Sample: 1. 3-Hydroxycotinine 2. Nornicotine 3. Cotinine 4. Anabasine 5. Nicotine ESI Ionization Source Parameters: Curtain Gas (CUR): 20 Collision Gas (CAD): 7 Temperature (TEM): 600 Gas 1 (GS1): 50 Gas 2 (GS2): 50 IS: 4500 Entrance Potential (EP): 10 Collision Potential (CXP): 12 Ann Montross Technical Specialist Ann enjoys cooking/baking and roasting her own coffee beans. Sample Pretreatment Equipment and Materials Standards
Transcript of TN-0103 APPLICATIONS · TN-0103 For additional technical notes, visit Page 1 of 8 APPLICATIONS A...
TN-0103
For additional technical notes, visit www.phenomenex.com Page 1 of 8
APPLICATIONS
A Sensitive Extraction Method for Nicotine and Metabolites from Urine using Simplified Liquid Extraction (SLE)Xianrong (Jenny) Wei, Ann Montross, and Sean OrlowiczPhenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
SLE ConditionsLoad: Load pretreated sample onto the Novum MAX 96-Well Plate (Phenomenex Part No.: 8E-S138-5GA) and apply 5” Hg of vacuum to initiate loading. Stop vacuum and wait 5 minutes.
Elute: Dispense 600 µL of 1 % Formic acid in DCM/IPA (95:5). Re-peat two more times. Collect eluents in a collection plate (2 mL).
Dry Down: Evaporate the final extract to complete dryness under a slow stream of N2 at 45 °C.
Reconstitute: Add 300 µL of 20 mM Ammonium Bicarbonate, pH 8.2/Methanol (90:10) and vortex.
This method has been validated and shown acceptable accuracy and precision using a small volume of human urine in the analy-sis of nicotine and its metabolites (anabasine, nornicotine, cotinine and 3-hydroxycotinine). The modified method employed uses a Novum™ SLE MAX 96-well plate with pH adjusted organic elution solvent, and a Kinetex® EVO C18 column with high pH mobile phase to increase analyte recoveries and sensitivity using LC/MS/MS.
IntroductionAnalysis of nicotine in urine has become more widespread with the increased use of tobacco-less products, such as nicotine lozenges, vaporizers, and gums. Analyzing nicotine metabolites (anabasine, nornicotine, cotinine and 3-hydroxycotinine) allows researchers to distinguish between an active smoker and some-one using tobacco-less products. The presence of tobacco me-tabolites, anabasine and nornicotine, in urine is an indication of an active smoker.
The goals of this study were to simplify the extraction process using a Simplified Liquid Extraction (SLE) method, increase re-coveries, and demonstrate the assay accuracy, precision and lin-earity for all compounds under (GLP) Good Laboratory Pratice guidance. The endogenous level of nicotine and its metabolites were monitored during the method development for the deter-mination of LLOQ quantification level purpose. The method uses higher pH mobile phase at pH 8.2 to ensure the separations of all compounds, and therefore the Kinetex EVO C18 column was chosen for its widely stable pH range and chromatography.
Experimental ConditionsReagents and ChemicalsAll solvents and reagents were HPLC or analytical grade. HPLC Grade methanol was purchased from Honeywell Burdick & Jackson® (Muskegon, MI), Milli-Q® water was used for sample preparation. HPLC Grade water was purchased from Honeywell Burdick & Jackson and used to prepare the LC mobile phase.
Reference standards were purchased from Cerilliant® Corporation.
Agilent® 1260 pumps and autosampler were used along with a SCIEX QTRAP® 4500, positive polarity, ESI for detection.
200 µL of 2 % NH4OH in water and 50 µL of mixed deuterated in-ternal standards (200 ng/mL each in water) were added to 100 µL of human urine (spiked with analytes and/or blank) and mixed.
LC/MS/MS Conditions: Column: Kinetex® 2.6 µm EVO C18
Dimensions: 100 x 3.0 mmPart No.: 00D-4725-Y0
Mobile Phase: A: 20 mM Ammonium Bicarbonate, pH 8.2B: Methanol
Gradient: Time (min) B (%)0 103 905 905.01 106. 10
Flow Rate: 0.75 mL/minTemperature: Ambient
Detection: SCIEX QTRAP 4500 Turbo™
Sample: 1. 3-Hydroxycotinine2. Nornicotine3. Cotinine4. Anabasine5. Nicotine
ESI Ionization Source Parameters:Curtain Gas (CUR): 20
Collision Gas (CAD): 7 Temperature (TEM): 600
Gas 1 (GS1): 50Gas 2 (GS2): 50
IS: 4500Entrance Potential (EP): 10
Collision Potential (CXP): 12
Ann MontrossTechnical SpecialistAnn enjoys cooking/baking and roasting her own coffee beans.
Sample Pretreatment
Equipment and Materials
Standards
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Table 1. Mass Transitions
Compounds Q1 Mass (amu)
Q3 Mass (amu)
Dwell Time (amu) DP CE
Anabasine 1* 163 80 25 80 27
Anabasine 2 163 120.1 25 80 22
Anabasine D4 1* 167 84 25 80 27
Anabasine D4 2 167 124 25 80 22
Cotinine 1 177 80 25 80 29
Cotinine 2* 177 98 25 70 28
Cotinine D3 1* 180 80 25 80 28
Cotinine D3 2 180 101 25 80 28
3-Hydroxycotinine 1* 193 80 25 60 34
3-Hydroxycotinine 2 193 134 25 60 25
3-Hydorxycotinine D3 1* 196 80 25 60 34
3-Hydroxycotinine D3 2 196 134 25 60 25
Nicotine 1 163 130 25 60 26
Nicotine 2* 163 132 25 60 20
Nicotine D4 1* 167 134 25 60 26
Nicotine D4 2 167 136 25 60 20
Nornicotine 1 149 80 25 50 25
Nornicotine 2* 149 130 25 50 21
Nornicotine D4 1 153 84 25 55 29
Nornicotine D4 2* 153 134 25 55 18
Anabasine 1* 163 80 25 80 27
* Quantitation mass
Table 2. Novum Recovery
Figure 1. Representative chromatograms of QCM at 200 ng/mL in human urine
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Table 1. Mass Transitions
Compounds Q1 Mass(amu)
Q3 Mass(amu)
Dwell Time(amu) DP CE
Anabasine 1* 163 80 25 80 27
Anabasine 2 163 120.1 25 80 22
Anabasine D4 1* 167 84 25 80 27
Anabasine D4 2 167 124 25 80 22
Cotinine 1 177 80 25 80 29
Cotinine 2* 177 98 25 70 28
Cotinine D3 1* 180 80 25 80 28
Cotinine D3 2 180 101 25 80 28
3-Hydroxycotinine 1* 193 80 25 60 34
3-Hydroxycotinine 2 193 134 25 60 25
3-Hydorxycotinine D3 1* 196 80 25 60 34
3-Hydroxycotinine D3 2 196 134 25 60 25
Nicotine 1 163 130 25 60 26
Nicotine 2* 163 132 25 60 20
Nicotine D4 1* 167 134 25 60 26
Nicotine D4 2 167 136 25 60 20
Nornicotine 1 149 80 25 50 25
Nornicotine 2* 149 130 25 50 21
Nornicotine D4 1 153 84 25 55 29
Nornicotine D4 2* 153 134 25 55 18
Anabasine 1* 163 80 25 80 27
* Quantitation mass
Table 2. Novum Recovery
Anabasine Nicotine Nornicotine Cotinine 3-OH-Cotinine
QCL 71.1 82.8 62.4 96.2 73.0
QCM 79.5 83.9 64.3 95.6 77.5
QCH 83.5 89.6 70.4 91.0 76.7
Figure 1. Representative chromatograms of QCM at 200ng/mL in human urine
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EN
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PS
Ap
p ID
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87
1. 3-Hydroxycotinine
2. Nornicotine
3. Cotinine
4. Anabasine
5. Nicotine
1. 3-Hydroxycotinine
2. Nornicotine
3. Cotinine
4. Anabasine
5. Nicotine
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p ID
237
87
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Anabasine Nicotine Nornicotine Cotinine 3-OH-ConitineQCL 71.1 82.8 62.4 96.2 73.0
QCM 79.5 83.9 64.3 95.6 77.5
QCH 83.5 89.6 70.4 91.0 76.7
Anabasine Nicotine Nornicotine Cotinine 3-OH-Cotinine
QCL 71.1 82.8 62.4 96.2 73.0
QCM 79.5 83.9 64.3 95.6 77.5
QCH 83.5 89.6 70.4 91.0 76.7
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Table 3. Accuracy and Precision
Anabasine Nicotine Nornicotine Cotinine 3-OH-Cotinine Sample ID
Nominal Conc. (ng/mL) 1 5 1 1 1LLOQ_1 0.946 5.14 1.15 1.05 0.979
LLOQ_2 0.950 4.45 0.994 0.900 0.900
LLOQ_3 0.993 4.27 1.04 0.941 0.987
LLOQ_4 0.986 6.56 0.942 0.971 0.988
LLOQ_5 0.962 5.29 1.02 1.08 1.18
LLOQ_6 1.09 5.32 1.15 1.04 1.10
Mean Conc. Found (ng/mL) 0.988 5.172 1.05 0.997 1.02
STDV 0.054 0.812 0.085 0.070 0.10
CV% 5.42 15.7 8.06 7.06 9.80
Accuracy (%) 98.8 103 105 99.7 102
n= 6 6 6 6 6
Sample IDNominal Conc. (ng/mL) 5 15 5 5 5
QCL_1 5.71 17.0 5.12 5.49 5.53
QCL_2 5.20 17.2 4.90 4.82 5.06
QCL_3 4.70 15.8 4.91 5.32 5.75
QCL_4 5.43 17.1 5.27 5.38 4.90
QCL_5 5.47 15.4 5.08 5.56 4.57
QCL_6 5.50 16.8 5.22 5.57 5.04
Mean Conc. Found (ng/mL) 5.34 16.55 5.08 5.36 5.14
STDV 0.351 0.758 0.154 0.281 0.430
CV% 6.58 4.58 3.03 5.24 8.36
Accuracy (%) 107 110 102 107 103
n= 6 6 6 6 6
Sample IDNominal Conc. (ng/mL) 200 200 200 200 200
QCM_1 213 210 213 197 222
QCM_2 221 211 202 198 194
QCM_3 225 203 207 222 215
QCM_4 212 214 206 224 213
QCM_5 214 224 222 209 217
QCM_6 234 222 204 231 218
Mean Conc. Found (ng/mL) 220 214 209 214 213
STDV 8.61 7.87 7.38 14.29 9.87
CV% 3.92 3.68 3.53 6.69 4.63
Accuracy (%) 110 107 105 107 107
n= 6 6 6 6 6
Sample IDNominal Conc. (ng/mL) 400 400 400 400 400
QCH_1 413 414 409 395 393
QCH_2 370 396 411 367 399
QCH_3 402 397 385 378 387
QCH_4 421 438 390 395 413
QCH_5 381 392 407 417 396
QCH_6 378 377 410 371 391
Mean Conc. Found (ng/mL) 394 402 402 387 397
STDV 20.8 21.1 11.4 18.8 9.1
CV% 5.27 5.24 2.84 4.85 2.29
Accuracy (%) 98.5 101 101 96.8 99.1
n= 6 6 6 6 6
Conc. Found (ng/mL) - QCL
Conc. Found (ng/mL) - QCM
Conc. Found (ng/mL) - QCH
Conc. Found (ng/mL) - LLOQ
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Figure 2. Representative chromatograms of blank human urine
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Figure 2. Representative chromatograms of blank human urine
Figure 3. Representative chromatogram of LLOQ at 1.0 ng/mL in human urine
Ap
p ID
237
84
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a) Anabasine
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b) Nicotine
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p ID
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85
Figure 3. Representative chromatogram of LLOQ at 1.0 ng/mL (5.0 ng/mL for Nicotine) in human urine
Ap
p ID
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84
a) Anabasine
d) Cotinine
d) Cotinine
b) Nicotine
b) Nicotine
e) 3-Hydroxycotinine
c) Nornicotine
c) Nornicotine
Ap
p ID
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85
a) Anabasine
e) 3-Hydroxycotinine
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Figure 4. Representative chromatogram of ULOQ at 500ng/mL in human urine
Figure 5. Representative calibration curves in human urine (n=2)
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500Analyte Conc. / IS Conc.
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Anabasine: 1 - 500 ng/mL
r = 0.9988
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c) Nornicotine
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Nornicotine: 1 - 500 ng/mL
r = 0.9996
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Cotinine: 1 - 500 ng/mL
r = 0.9993
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r = 0.9991
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Nicotine: 5 - 500 ng/mL
r = 0.9990
Ap
p ID
: 237
86
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Figure 2. Representative chromatograms of blank human urine
Figure 3. Representative chromatogram of LLOQ at 1.0ng/mL in human urine
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84
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Inte
nsity
, cp
s
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
Inte
nsity
, cp
s
a) Anabasine
a) Anabasine
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
1.05e4
1.10e4
1.15e4
1.20e41.24e4
Inte
nsity
, cp
s
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
Inte
nsity
, cp
s
d) Cotinine
d) Cotinine
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0min 0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
Inte
nsity
, cp
s
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e43.5e4
Inte
nsity
, cp
s
b) Nicotine
b) Nicotine
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
Inte
nsity
, cp
s
Max. 3.0e4cps.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
Inte
nsity
, cp
s
e) 3-Hydroxycotinine
e) 3-Hydroxycotinine
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
Inte
nsity
, cp
s
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
Inte
nsity
, cp
s
c) Nornicotine
c) Nornicotine
Ap
p ID
237
85
Figure 4. Representative chromatogram of ULOQ at 500 ng/mL in human urine
Figure 5. Representative calibration curves in human urine (n=2)
Anabasine: 1 - 500 ng/mL
r = 0.9988
d) Cotinine e) 3-Hydroxycotinine
c) Nornicotine
Nornicotine: 1 - 500 ng/mL
r = 0.9996
Cotinine: 1 - 500 ng/mL
r = 0.9993
3-Hydroxycotinine: 1 - 500 ng/mL
r = 0.9991
Nicotine: 5 - 500 ng/mL
r = 0.9990
Ap
p ID
: 237
86
b) Nicotinea) Anabasine
TN-0103
Page 6 of 8
Results and DiscussionThe assay of nicotine and metabolites (anabasine, nornicotine, cotinine and 3-hydroxycotinine) was validated for linearity, accu-racy and precision using five levels of calibration standards for all compounds. This method was able to obtain the LLOQ at 1 ng/mL (5 ng/mL for Nicotine) using SCIEX QTRAP® 4500. Also with the modifications to the SLE method with improved recovery of ana-lytes, the need of sample volume was reduced to 100 µL making this method ideal for any laboratory environment.
There were several modifications made to the SLE extraction that produced the higher recoveries over the entire calibration range. The addition of 2 % ammonium hydroxide neutralized the analytes before the sample loading, and the 1 % formic acid added to the elution step were the two major modifications that increased re-coveries as seen in Table 2. The mass transitions of the analytes were listed in Table 1, respectively. The optimal mobile phase re-quired 20 mM ammonium bicarbonate with a pH of 8.2 to ensure
separation and peak shape of analysis. A representative total ion chromatogram at medium QC level (200 ng/mL) is shown in Fig-ure 1. A representative chromatogram of a blank, Lowest Limit of Quantitation (LLOQ), and Upper Limit of Quantitation (ULOQ) are seen in Figures 2, 3, and 4 respectively.
The results of the accuracy and precision from the LLOQ, Quality Control Low (QCL), Quality Control Medium (QCM), and Quality Control High (QCH) meet the GLP acceptance criteria (Table 3). The assay accuracy at above four QC levels over all compounds was between 96.8 and 110 %. The Coefficient of Variation (% CV) for the LLOQ ranged between 5.42 % and 15.7 %; the QCLanalytes had a % CV that ranged between 3.03 % and 8.36 %; the QCM analytes had a % CV that ranged between 3.53 % and 6.69 %; the QCH analytes had a % CV that ranged between 2.29 % and 5.27 %, respectively. The linearity of each calibration curve had the following R2 values from 0.9988 to 0.9996 for all analytes. The dynamic curve ranges are at 1-500 ng/mL for all compounds except Nicotine at 5-500 ng/mL due to the endoge-nous level of nicotine in human body (Figure 2 and 5).
The internal standard responses were also evaluated. The assay showed the consistent response of internal standard, which was within ±20 % of mean of standards and QCs in the run for all com-pounds (Figure 6).
ConclusionThe simplified quantitation method of analysis of nicotine, anab-asine, nornicotine, cotinine and 3-hydroxycotinine in human urine shows acceptable, accurate, and precise results over the wide calibration range. The modified SLE method was optimized to provide higher recovery across all compounds. The Kinetex® EVO C18 column coupled with high pH mobile phase showed better peak shape, resolution and sensitivity in the analysis of nicotine and its metabolites in human urine. The lower sample volume and optimized simplified liquid extraction method can be easily transfer into various laboratory settings. The assay is also auto-mation friendly which allows for high throughput laboratories to utilize this method.
5 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk00A-4633-AN 00B-4633-AN 00D-4633-AN 00F-4633-AN AJ0-9298
for 2.1 mm ID
5 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 3.0 100 x 3.0 150 x 3.0 3/pk00B-4633-Y0 00D-4633-Y0 00F-4633-Y0 AJ0-9297
for 3.0 mm ID
5 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk00B-4633-E0 00D-4633-E0 00F-4633-E0 00G-4633-E0 AJ0-9296
for 4.6 mm ID
Kinetex EVO C18 Core-Shell LC ColumnsOrdering Information
2.6 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk00A-4725-AN 00B-4725-AN 00D-4725-AN 00F-4725-AN AJ0-9298
for 2.1 mm ID
2.6 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 3.0 100 x 3.0 150 x 3.0 3/pk00B-4725-Y0 00D-4725-Y0 00F-4725-Y0 AJ0-9297
for 3.0 mm ID
2.6 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 4.6 100 x 4.6 150 x 4.6 3/pk00B-4725-E0 00D-4725-E0 00F-4725-E0 AJ0-9296
for 4.6 mm ID
1.7 μm Minibore Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 2.1 100 x 2.1 150 x 2.1 3/pk00B-4726-AN 00D-4726-AN 00F-4726-AN AJ0-9298
for 2.1 mm ID‡ SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000
TN-0103
Page 6 of 8
Results and DiscussionThe assay of nicotine and metabolites (anabasine, nornicotine,cotinine and 3-hydroxycotinine) was validated for linearity, accu-racy and precision using five levels of calibration standards for allcompounds. This method was able to obtain the LLOQ at 1 ng/mL(5 ng/mL for Nicotine) using SCIEX QTRAP® 4500. Also with themodifications to the SLE method with improved recovery of ana-lytes, the need of sample volume was reduced to 100 µL makingthis method ideal for any laboratory environment.
There were several modifications made to the SLE extraction thatproduced the higher recoveries over the entire calibration range.The addition of 2 % ammonium hydroxide neutralized the analytesbefore the sample loading, and the 1 % formic acid added to theelution step were the two major modifications that increased re-coveries as seen in Table 2. The mass transitions of the analyteswere listed in Table 1, respectively. The optimal mobile phase re-quired 20 mM ammonium bicarbonate with a pH of 8.2 to ensure
separation and peak shape of analysis. A representative total ionchromatogram at medium QC level (200 ng/mL) is shown in Fig-ure 1. A representative chromatogram of a blank, Lowest Limit ofQuantitation (LLOQ), and Upper Limit of Quantitation (ULOQ) areseen in Figures 2, 3, and 4 respectively.
The results of the accuracy and precision from the LLOQ, QualityControl Low (QCL), Quality Control Medium (QCM), and QualityControl High (QCH) meet the GLP acceptance criteria (Table 3). The assay accuracy at above four QC levels over all compoundswas between 96.8 and 110 %. The Coefficient of Variation (%CV) for the LLOQ ranged between 5.42 % and 15.7 %; the QCLanalytes had a % CV that ranged between 3.03 % and 8.36 %; the QCM analytes had a % CV that ranged between 3.53 % and6.69 %; the QCH analytes had a % CV that ranged between2.29 % and 5.27 %, respectively. The linearity of each calibrationcurve had the following R2 values from 0.9988 to 0.9996 for allanalytes. The dynamic curve ranges are at 1-500 ng/mL for allcompounds except Nicotine at 5-500 ng/mL due to the endoge-nous level of nicotine in human body (Figure 2 and 5).
The internal standard responses were also evaluated. The assayshowed the consistent response of internal standard, which waswithin ±20 % of mean of standards and QCs in the run for all com-pounds (Figure 6).
ConclusionThe simplified quantitation method of analysis of nicotine, anab-asine, nornicotine, cotinine and 3-hydroxycotinine in human urineshows acceptable, accurate, and precise results over the widecalibration range. The modified SLE method was optimized toprovide higher recovery across all compounds. The Kinetex® EVOC18 column coupled with high pH mobile phase showed betterpeak shape, resolution and sensitivity in the analysis of nicotineand its metabolites in human urine. The lower sample volumeand optimized simplified liquid extraction method can be easilytransfer into various laboratory settings. The assay is also auto-mation friendly which allows for high throughput laboratories toutilize this method.
Figure 6. Representative Internal Standard Plots
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340Index
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.1e6
IS P
eak
Are
a, c
ount
s
Ap
p ID
237
88
Cotinine
5 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk00A-4633-AN 00B-4633-AN 00D-4633-AN 00F-4633-AN AJ0-9298
for 2.1 mm ID
5 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 3.0 100 x 3.0 150 x 3.0 3/pk00B-4633-Y0 00D-4633-Y0 00F-4633-Y0 AJ0-9297
for 3.0 mm ID
5 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk00B-4633-E0 00D-4633-E0 00F-4633-E0 00G-4633-E0 AJ0-9296
for 4.6 mm ID
Kinetex EVO C18 Core-Shell LC ColumnsOrdering Information
2.6 μm Minibore Columns (mm)SecurityGuard™
ULTRA Cartridges‡
30 x 2.1 50 x 2.1 100 x 2.1 150 x 2.1 3/pk00A-4725-AN 00B-4725-AN 00D-4725-AN 00F-4725-AN AJ0-9298
for 2.1 mm ID
2.6 μm MidBore™ Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 3.0 100 x 3.0 150 x 3.0 3/pk00B-4725-Y0 00D-4725-Y0 00F-4725-Y0 AJ0-9297
for 3.0 mm ID
2.6 μm Analytical Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 4.6 100 x 4.6 150 x 4.6 3/pk00B-4725-E0 00D-4725-E0 00F-4725-E0 AJ0-9296
for 4.6 mm ID
1.7 μm Minibore Columns (mm)SecurityGuard
ULTRA Cartridges‡
50 x 2.1 100 x 2.1 150 x 2.1 3/pk00B-4726-AN 00D-4726-AN 00F-4726-AN AJ0-9298
for 2.1 mm ID‡ SecurityGuard ULTRA Cartridges require holder, Part No.: AJ0-9000
± 20 % Mean of all samples
Figure 6. Representative Internal Standard Plots
Ap
p ID
237
88
Cotinine
TN-0103
For additional technical notes, visit www.phenomenex.com Page 7 of 8
If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 45 days for a FULL REFUND.
96-Well Plate Accessories Part No. Description UnitCollection Plates (deep well, polypropylene)AH0-7192 96-Well Collection Plate, 350 µL/well 50/pkAH0-7193 96-Well Collection Plate, 1 mL/well 50/pkAH0-7194 96-Well Collection Plate, 2 mL/well 50/pkAH0-8635 96-Well Collection Plate, 2 mL/well Square/Round-Conical 50/pkAH0-8636 96-Well Collection Plate, 2 mL/well Round/Round, 8 mm 50/pkAH0-7279 96-Well Collection Plate, 1 mL/well Round, 7 mm 50/pk
Sealing MatsAH0-8597 Sealing Mats, Pierceable, 96-Square Well, Silicone 50/pkAH0-8598 Sealing Mats, Pre-Slit, 96-Square Well, Silicone 50/pkAH0-8631 Sealing Mats, Pierceable, 96-Round Well 7 mm, Silicone 50/pkAH0-8632 Sealing Mats, Pre-Slit, 96-Round Well 7 mm, Silicone 50/pkAH0-8633 Sealing Mats, Pierceable, 96-Round Well 8 mm, Silicone 50/pkAH0-8634 Sealing Mats, Pre-Slit, 96-Round Well 8 mm, Silicone 50/pkAH0-7362 Sealing Tape Pad 10/pk
Vacuum ManifoldAH0-8950 96-Well Plate Manifold, Universal with Vacuum Gauge ea
Novum™ Simplified Liquid Extraction (SLE) 96-Well Plates Part No. Description Unit8E-S138-FGA Novum SLE MINI 96-Well Plate 1/pk
8E-S138-5GA Novum SLE MAX 96-Well Plate 1/pk
Novum SLE Tubes Part No. Description Unit
8B-S138-FAK Novum SLE 1 cc tubes 100/pk
8B-S138-5BJ Novum SLE 3 cc tubes 50/pk
8B-S138-JCH Novum SLE 6 cc tubes 30/pk
8B-S138-KDG Novum SLE 12 cc tubes 20/pk
Ordering InformationVacuum Manifolds
Part No. Description Unit
12–Position Vacuum Manifold for Tubes*
AH0-6023 12-Position Vacuum Manifold Set, complete assembly ea
24–Position Vacuum Manifold for Tubes*
AH0-6024 24-Position Vacuum Manifold Set, complete assembly ea
96-Well Plate Manifold
AH0-8950 96-Well Plate Manifold, Universal with vacuum gauge ea
* Manifolds include: Vacuum-tight glass chamber, vacuum gauge assembly, polypropylene lid withzgasket, male and female luers and yellow end plugs, stopcock valves, collection rack assemblies, polypropylene needles, lid support legs. Waste container included with 12-position manifold.
Presston 100 Tube Adapter Kits (for AH0-9334)Part No. Description
AH0-9344 1 mL Tube Adapter Kit
AH0-9345 3 mL Tube Adapter Kit
AH0-9346 6 mL Tube Adapter Kit
Presston™ 100 Positive Pressure ManifoldPart No. Description
AH0-9334 Presston 100 Positive Pressure Manifold, 96-Well Plate
AH0-9342 Presston 100 Positive Pressure Manifold, 1 mL Tube Complete Assembly
AH0-9347 Presston 100 Positive Pressure Manifold, 3 mL Tube Complete Assembly
AH0-9343 Presston 100 Positive Pressure Manifold, 6 mL Tube Complete Assembly
The Presston 100 96-Well Positive Pressure Manifold can also process 1, 3, and 6 mL tubes using the following adapter kits
WARRANTY Phenomenex warrants that for a period of 12 months following delivery, the Presston 100 Positive Pressure Manifold you have purchased will perform in accordance with the published specifications and will be free from defects in materials or workmanship. In the event that the Presston 100 Positive Pressure Manifold does not meet this warranty, Phenomenex will repair or replace defective parts. Please visit www.phenomenex.com/Presston for complete warranty information.
TN-0103
Page 8 of 8
APPLICATIONS
TN45
3410
16_W
Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions.
Trademarks Kinetex is a registered trademark and Novum, Presston, MidBore, and SecurityGuard are trademarks of Phenomenex. Milli-Q is a registered trademark of MERCK KGaA, Darmstandt, Germany. Agilent is a registered trademark of Agilent Technologies, Inc. QTRAP is a registered trademark of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license. Cerilliant is a registered trademark of Cerilliant Corporation. Burdick & Jackson is a registered trademark of Honeywell International Inc.
© 2016 Phenomenex, Inc. All rights reserved.
www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected]
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