APPLICATION OF UPLC OPEN ARCHITECTURE … · use of Ayahuasca and other “plant teachers” for...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2009 Waters Corporation | COMPANY CONFIDENTIAL METHODS 1. Harmalol 2. Harmalol Assoc. Peak 3. Norharmane 4. Harmaline 5. Harmane 6. Harmine DMT Determination of optimal ex λ and em λ INTRODUCTION Figure 2: Waters UPLC® Open Architecture System CONCLUSIONS • Significant solvent savings are achieved. • HPLC requires 24mL solvent per analysis vs. 3.25mL for UPLC . UPLC assay uses methanol! • Run time reduced from 20 minutes to 6.5 minutes • Original HPLC assays required two separate analysis for the detection and quantification of harmanes(HPLC) and DMT (GC). The new UPLC allows for identification and quantification of both harmanes and DMT in the same analysis. • IntelliStart ™ technology automates development of MS/ MS method for optimum detection and identification of compounds of interest. Great for the new user! • ACQUITY FLR 3D scan function simplifies determination of optimal excitation and emission wavelength. • ACQUITY Open Architecture allows use of either the AC- QUITY Sample Manage or the 2777C with it’s many poten- tial capabilities for on - line sample preparation . RESULTS Figure 7. (left). 1.0 mcg/mL Standard material = 50ng on column. 1. Harmalol MRM 200.1>172.0 2. Harmalol Assoc peak 3. Norharmane MRM 169.1>115.1 4. Harmaline MRM 215.1>174.1 5. Harmane MRM 183.1>142.1 6. Harmine MRM213.1>169.9 7. DMT MRM 189.1 >58.0 PDA MAX PLOT 240NM TO 350NM FLR EX λ309 EM 409λ Table 2. Below. Limit of detection for each compound expressed as ng on column. limit of detection = ng on column channel Harmalol Norharmane Harmaline Harmane Harmine DMT FLR ND 50 ND 25 25 PDA 12.5 12.5 25 12.5 12.5 ND 200.1>172.0 12.5 169.1>115.1 7 215.1>174.1 0.25 183.1>142.1 0.25 213.1>169.9 8 189.1>58.0 0.6 Figure 9: 10 ng to 1000ng/mL calibration curve for harmaline. Calibration curve R 2 for all compounds was 0.992 or better across the dual calibration ranges from 10 to 1000 and 100 to 10,000 ng/mL. Table 1: Quantitative results for brewed tea, leaf extract and vine extract. 1. Clinical investigations of the therapeutic potential of ayahuasca:. McKenna, Dennis J. 2004, Pharmacology & Therapeutics , pp. 111- 129. 2. Determination of N,N-dimethyltryptamine and b-carboline alkaloids in human plasma following oral administration of Ayahuasca. Mercedes Yritiaa, Jordi Ribaa, Jordi Ortun˜ob, Ariel Ramirezb, Araceli Cas- tilloa,. 2002, Journal of Chromatography B,779, pp. 271–281. 3. Phytochemical Analyses of Banisteriopsis Caapi and Psychotria Viridis. J.C. Callaway, Ph.D.* and Glacus S. Brito, M.D.** & Edison S. Neves, M.D.**. 2, June 2005, Journal of Psychoactive Drugs, Vol. Vol- ume 37, pp. 145 - 150. 4. Fast, Easy and Efficient Ultra Performance LC Method Development. Waters p/n: 720001806EN. Doug McCabe, 2006. Authors gratefully acknowledge Andrew Aubin and Claude Mallet of Waters Chromatography for their contributions to this work. concentration ng/mL sample Harmalol Norhar‐ mane Harmaline Harmane Harmine DMT Brewed tea 632 ND 1146 26 9,774 20 Leaf extract 1230 268 40 2 ND 22 Vine extract 6000 272 9927 2084 17,987 180 1 2 4 5 6 AU 0.00 0.60 1.20 2 6 EU 0.00 5.00 10.00 1 2 Intensity 0 200000 400000 4 Intensity 0 4x10 6 8x10 6 5 Intensity 0.0 15000.0 30000.0 6 Intensity 0.0 1.5x10 7 3.0x10 7 Intensity 0.0 40000.0 80000.0 7 Intensity 2600.0 5200.0 7800.0 Minutes 1.80 2.70 3.60 4.50 5.40 Figure 8. Brewed Ayahuasca tea. Channel designations are identical to those shown for figure 7. 1 2 3 4 5 6 AU 0.00 0.02 2 3 5 6 EU 0.00 0.10 1 2 Intensity 0.0 20000.0 4 Intensity 0 900000 5 Intensity 0 700000 6 Intensity 0 2x10 6 3 Intensity 0 5x10 6 7 Intensity 0 3x10 6 Minutes 1.80 2.70 3.60 4.50 5.40 Abstract Number: 870 - 6P; Session 870 - Biomedical Analysis and Synthesis; Day and Time: Monday, March 09, 2009, Afternoon Figure 1:left to right. Banisteriopsis caapi, Psychotria viridis, Ayahuasca cooking in Ecuador This poster describes the transfer and subsequent optimization of an HPLC/Fluorescence (FLR) method for analysis of harmala alkaloids (2),(3) to Ultra Performance LC ® analysis using the UPLC Open Architecture System with ACQUITY PDA , Fluorescence and TQD detectors for the identification and quantification of harmala alkaloids in Ayahuasca tea and samples of Banisteriopsis caapi and Psychotria viridis. Banisteriopsis caapi, also known as Ayahuasca, Caapi or Yage, is a South American jungle vine of the family Malpighiaceae. B. caapi is most often used in combination with the Psychotria viridis plant to prepare Ayahuasca, a decoction considered a sacred medicine among the indigenous peoples of the Amazon Rainforest. The use of Ayahuasca and other “plant teachers” for visionary experiences and healing purposes is an ancient practice which has attracted the attention of modern psychiatry for its potential as a treatment for depression and other psychiatric disorders(1). Ayahuasca, also known as Hoasca or “vine of the souls” by the Quechua Indians contains the beta-carboline harmala alkaloids and MAOIs harmine, harmaline, and tetrahydroharmine. The MAOIs in B. caapi allow the primary psychoactive compound, N,N- dimethyltryptamine (which is introduced from the Psychotria viridis plant), to be orally active. The chemical nature of its active constituents and the manner of its use makes Ayahuasca study relevant to contemporary issues in neuropharmacology, neurophysiology, and psychiatry. This interest supports continued investment and improvement in analytical methodology required for study of the active constituents of Ayahuasca teas. APPLICATION OF UPLC ® OPEN ARCHITECTURE SYSTEM WITH PDA, FLR & MS/MS DETECTION TO THE DETERMINATION OF ß-CARBOLINE ALKALOID CONTENT IN AYAHUASCA TEA AND SOURCE PLANTS BANISTERIOPSIS CAAPI AND PSYCHOTRIA VIRIDIS Authors: Patricia McConville , Charles Phoebe, Ph.D. and Daniel Root, Ph.D Affiliations: Waters Corporation, 34 Maple Street. Milford MA USA Reference Materials: Harmane, harmaline, harmalol, norharmalol and harmine reference materials were obtained from ChromaDex™. 10005 Muirlands Blvd. Suite G, First floor. Irvine, CA. www.chromadex.com . 1mg of N,N-Dimethyltryptamine (DMT) in 1mL of methanol was obtained from Alltech. Alltech Applied Science. 2701 Carolean Drive. State College, PA. 16801. www.discoverysciences.com . Banisteriopsis caapi (114g of powdered vine) and 28g of dried foliage of the Psychotria viridis plant were obtained from Bouncing Bear Botanicals. www.bouncingbearbotanicals.com Sample Preparation: Ayahuasca tea: 5g of powdered banisteriopsis caapi vine, 2.5g of the dried Psychotria viridis foliage and 1 L of MilliQ water were placed into a 2L Pyrex beaker and heated with stirring to a gentle boil. The mixture was allowed to simmer for 2 hours, reducing the liquid to approximately 0.5L. The resulting mixture was filtered with Grade No. 1 Filter Paper, (Whatman) and frozen at -20°C until analysis. The tea was brought to room temperature and filtered through Acrodisc® CR 13mm, 0.2μm into a sample vial. 5μL injected. Leaf and vine extracts: 5 grams of Psychotria viridis foliage was subjected to the Soxhlate Extraction method with 150 mLs hot methanol. 5g of powdered banisteriopsis caapi vine was subjected to Soxhlate extraction method with 150mL of hot methanol. The methanolic extracts were stored at -20°C until analysis. Immediate prior to analysis the Soxhlate extracts were diluted 2x with MilliQ water then filtered through Acrodisc® CR 13mm, 0.2μm, into a sample vial. 5μL or 1μL injected. Working Calibration Standards: Individual stock solutions containing 1mg/mL of each drug were prepared in methanol and stored at -20°C. Calibration curves were constructed from combined working standards prepared in MilliQ water at concentrations from 0.01 mcg/mL to 0.1 mcg/mL and 0.1mcg/mL to 10 mcg/mL . Calibration solutions were stored protected from light at 10°C for up to 4 hours prior to injection. 5μL injected. Chromatographic Conditions: LC Scouting & Optimization Working from the LC conditions reported in Ref. 2, buffers at pH 8 and pH 10 were selected as starting points. The ACQUITY BEH C18 phase was selected based upon comparisons to the original analytical column, a Kromasil 100 C18 , 5 μm, 150 x 4 mm I.D. made in the Waters Column Selectivity Chart . http://www.waters.com A matrix (Figure 3) was generated using the following method scouting conditions: ACQUITY® TQD, ACQUITY FLR Detector, ACQUITY PDA, Waters 2777c High-Pressure Sample Manager or ACQUITY Sample Manger, ACQUITY Column Manger, ACQUITY Binary Solvent Manager. Split flow configuration to TQ and PDA>FLR. Sample: 50 mcg/mL mixture in 50%methanol 50%water. Column: 2.1 x 50mm ACQUITY UPLC ® BEH C18 1.7μm Column Temp: 40°C Flow Rate: 0.7 mL/min Mobile Phase: A1: 10mM Ammonium Bicarbonate pH 10 A2: 10mM Ammonium Acetate pH 8.0 (pH 3 buffer is 2nd option in the normal scout protocol) B1: Acetonitrile (Optima) B2: Methanol (Optima) Gradient: 1—95% Organic in 10 minutes Inj Volume: 3μL of 50 mcg/mL mixed harmanes & DMT in water (10 μL loop) Weak Wash: 50/50 Water/Acetonitrile Strong Wash: 100% methanol 1% ammonium hydroxide ACQUITY PDA λ 210 to 500nm Rs: 1.2nm Date rate: 20pts. TC: Fast. Optimization of detection conditions for TQ: Figure 5: Top: IntelliStart™ automatically developed optimal TQD settings for analysis. Reference materials in 50% metha- nol & water under combined flow from reservoir and mobile phase were used to establish MRMs for each compound. UP to 4 Masses may be tuned for simultaneously. Bottom: Final MRM and MS1 Scan conditions generated by IntelliStart™ Compound Function Ion Mode Start (min) Stop (min) MRM Dwell (sec) Cone (V) CE (V) Harmalol MRM ES+ 1.50 4.00 200.1 > 172.0 0.075 60 20 Harmaline MRM ES+ 3.00 6.00 215.1>174.1 0.100 30 24 Harmane MRM ES+ 3.00 6.00 183.1>142.1 0.100 45 26 Harmine MRM ES+ 3.00 6.00 213.1>169.9 0.100 25 34 Norhar‐ mane MRM ES+ 3.00 6.00 169.1>115.1 0.100 60 32 DMT MRM ES+ 3.00 6.00 189.1 > 58.0 0.100 25 12 All MS1 SCAN ES+ 1.50 6.00 Mass Range: 70 to 230 Da; Scan Rate: 5000 Da/ sec 30 Source: 150° C; Desolvation: 350°C ; Desolvation gas: 600 L/Hr; Cone: 1 L/Hr. Figure 3: PDA 238nm. 10 min gradients 1 to 95% were evaluated with both buffers and both organic solvents. The combination of pH 10 buffer & methanol (bottom plot) gave the best separation. At this point, modeling software Dry- Lab (http://www.molnar-institut.com/cd/e/dlw/ drylab1.html ) was used to optimize the separation. Final separation shown in Figure 4. 1 2 3 4 5 6 % METHANOL 14.00 28.00 42.00 56.00 70.00 84.00 Minutes 1.40 2.80 4.20 5.60 7.00 Figure 6: ACQUITY FLR 3D stop flow scan function en- ables accurate determination of optimal ex & em λ. 3D scan for harmine is shown. Figure 4: Standard material. PDA Max Plot (240.0 to 350.0nm) and MRM 189.1>58 with % methanol overlay. Final Method Column:2.1 x 100mm ACQUITY UPLC ® BEH C18 1.7μm; Column Temp:55°C; Flow Rate:0.5 mL/min. A1: 10mM Ammonium Bicarbonate pH 10; B2: Methanol Gradient:14% - 44% B2 0 to 2.50 min, hold 2min, 44% to 85% B at 6.50 min. Hold 1min, return to 14% B. 720002988en
Transcript of APPLICATION OF UPLC OPEN ARCHITECTURE … · use of Ayahuasca and other “plant teachers” for...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2009 Waters Corporation | COMPANY CONFIDENTIAL
METHODS 1. Harmalol 2. Harmalol Assoc. Peak 3. Norharmane 4. Harmaline 5. Harmane 6. Harmine DMT
Determination of optimal ex λ and em λ
INTRODUCTION
Figure 2: Waters UPLC® Open Architecture System
CONCLUSIONS • Significant solvent savings are achieved.
• HPLC requires 24mL solvent per analysis vs. 3.25mL for UPLC . UPLC assay uses methanol!
• Run time reduced from 20 minutes to 6.5 minutes • Original HPLC assays required two separate analysis for
the detection and quantification of harmanes(HPLC) and DMT (GC). The new UPLC allows for identification and quantification of both harmanes and DMT in the same analysis.
• IntelliStart ™ technology automates development of MS/MS method for optimum detection and identification of compounds of interest. Great for the new user!
• ACQUITY FLR 3D scan function simplifies determination of optimal excitation and emission wavelength.
• ACQUITY Open Architecture allows use of either the AC-QUITY Sample Manage or the 2777C with it’s many poten-tial capabilities for on - line sample preparation .
RESULTS
Figure 7. (left). 1.0 mcg/mL Standard material = 50ng on column. 1. Harmalol MRM 200.1>172.0 2. Harmalol Assoc peak 3. Norharmane MRM 169.1>115.1 4. Harmaline MRM 215.1>174.1 5. Harmane MRM 183.1>142.1 6. Harmine MRM213.1>169.9 7. DMT MRM 189.1 >58.0 PDA MAX PLOT 240NM TO 350NM FLR EX λ309 EM 409λ Table 2. Below. Limit of detection for each compound expressed as ng on column.
limit of detection = ng on column
channel Harmalol Norharmane Harmaline Harmane Harmine DMT FLR ND 50 ND 25 25 PDA 12.5 12.5 25 12.5 12.5 ND 200.1>172.0 12.5 169.1>115.1 7 215.1>174.1 0.25 183.1>142.1 0.25 213.1>169.9 8 189.1>58.0 0.6
Figure 9: 10 ng to 1000ng/mL calibration curve for harmaline. Calibration curve R2 for all compounds was 0.992 or better across the dual calibration ranges from 10 to 1000 and 100 to 10,000 ng/mL.
Table 1: Quantitative results for brewed tea, leaf extract and vine extract.
1. Clinical investigations of the therapeutic potential of ayahuasca:. McKenna, Dennis J. 2004, Pharmacology & Therapeutics , pp. 111-129.
2. Determination of N,N-dimethyltryptamine and b-carboline alkaloids in human plasma following oral administration of Ayahuasca. Mercedes Yritiaa, Jordi Ribaa, Jordi Ortun˜ob, Ariel Ramirezb, Araceli Cas-tilloa,. 2002, Journal of Chromatography B,779, pp. 271–281.
3. Phytochemical Analyses of Banisteriopsis Caapi and Psychotria Viridis. J.C. Callaway, Ph.D.* and Glacus S. Brito, M.D.** & Edison S. Neves, M.D.**. 2, June 2005, Journal of Psychoactive Drugs, Vol. Vol-ume 37, pp. 145 - 150.
4. Fast, Easy and Efficient Ultra Performance LC Method Development. Waters p/n: 720001806EN. Doug McCabe, 2006.
Authors gratefully acknowledge Andrew Aubin and Claude Mallet of Waters Chromatography for their contributions to this work.
concentration ng/mL
sample Harmalol Norhar‐mane Harmaline Harmane Harmine DMT
Brewed tea 632 ND 1146 26 9,774 20
Leaf extract 1230 268 40 2 ND 22 Vine extract 6000 272 9927 2084 17,987 180
1 2 4 56
AU
0.000.601.20
2
6
EU
0.005.00
10.00
1
2
Inte
nsity
0200000400000
4
Inte
nsity
04x1068x106
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0.015000.030000.0
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Inte
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0.01.5x1073.0x107
Inte
nsity
0.040000.080000.0
7
Inte
nsity
2600.05200.07800.0
Minutes1.80 2.70 3.60 4.50 5.40
Figure 8. Brewed Ayahuasca tea. Channel designations are identical to those shown for figure 7.
1 2
3
45
6AU
0.00
0.02
2 3
5 6
EU
0.00
0.10
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Inte
nsity
0.0
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4
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nsity
0
900000
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0
700000
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Inte
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0
2x106
3
Inte
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5x106
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Minutes1.80 2.70 3.60 4.50 5.40
Abstract Number: 870 - 6P; Session 870 - Biomedical Analysis and Synthesis; Day and Time: Monday, March 09, 2009, Afternoon
Figure 1:left to right. Banisteriopsis caapi, Psychotria
viridis, Ayahuasca cooking in Ecuador
This poster describes the transfer and subsequent optimization of an HPLC/Fluorescence (FLR) method for analysis of harmala alkaloids (2),(3) to Ultra Performance LC® analysis using the UPLC Open Architecture System with ACQUITY PDA , Fluorescence and TQD detectors for the identification and quantification of harmala alkaloids in Ayahuasca tea and samples of Banisteriopsis caapi and Psychotria viridis.
Banisteriopsis caapi, also known as Ayahuasca, Caapi or Yage, is a South American jungle vine of the family Malpighiaceae. B. caapi is most often used in combination with the Psychotria viridis plant to prepare Ayahuasca, a decoction considered a sacred medicine among the indigenous peoples of the Amazon Rainforest. The use of Ayahuasca and other “plant teachers” for visionary experiences and healing purposes is an ancient practice which has attracted the attention of modern psychiatry for its potential as a treatment for depression and other psychiatric disorders(1). Ayahuasca, also known as Hoasca or “vine of the souls” by the Quechua Indians contains the beta-carboline harmala alkaloids and MAOIs harmine, harmaline, and tetrahydroharmine. The MAOIs in B. caapi allow the primary psychoactive compound, N,N-dimethyltryptamine (which is introduced from the Psychotria viridis plant), to be orally active.
The chemical nature of its active constituents and the manner of its use makes Ayahuasca study relevant to contemporary issues in neuropharmacology, neurophysiology, and psychiatry. This interest supports continued investment and improvement in analytical methodology required for study of the active constituents of Ayahuasca teas.
APPLICATION OF UPLC® OPEN ARCHITECTURE SYSTEM WITH PDA, FLR & MS/MS DETECTION TO THE DETERMINATION OF ß-CARBOLINE ALKALOID CONTENT IN AYAHUASCA TEA AND SOURCE PLANTS BANISTERIOPSIS CAAPI AND PSYCHOTRIA VIRIDIS
Authors: Patricia McConville, Charles Phoebe, Ph.D. and Daniel Root, Ph.D Affiliations: Waters Corporation, 34 Maple Street. Milford MA USA
Reference Materials:
Harmane, harmaline, harmalol, norharmalol and harmine reference materials were obtained from ChromaDex™. 10005 Muirlands Blvd. Suite G, First floor. Irvine, CA. www.chromadex.com. 1mg of N,N-Dimethyltryptamine (DMT) in 1mL of methanol was obtained from Alltech. Alltech Applied Science. 2701 Carolean Drive. State College, PA. 16801. www.discoverysciences.com. Banisteriopsis caapi (114g of powdered vine) and 28g of dried foliage of the Psychotria viridis plant were obtained from Bouncing Bear Botanicals. www.bouncingbearbotanicals.com Sample Preparation:
Ayahuasca tea: 5g of powdered banisteriopsis caapi vine, 2.5g of the dried Psychotria viridis foliage and 1 L of MilliQ water were placed into a 2L Pyrex beaker and heated with stirring to a gentle boil. The mixture was allowed to simmer for 2 hours, reducing the liquid to approximately 0.5L. The resulting mixture was filtered with Grade No. 1 Filter Paper, (Whatman) and frozen at -20°C until analysis. The tea was brought to room temperature and filtered through Acrodisc® CR 13mm, 0.2µm into a sample vial. 5µL injected. Leaf and vine extracts: 5 grams of Psychotria viridis foliage was subjected to the Soxhlate Extraction method with 150 mLs hot methanol. 5g of powdered banisteriopsis caapi vine was subjected to Soxhlate extraction method with 150mL of hot methanol. The methanolic extracts were stored at -20°C until analysis. Immediate prior to analysis the Soxhlate extracts were diluted 2x with MilliQ water then filtered through Acrodisc® CR 13mm, 0.2µm, into a sample vial. 5µL or 1µL injected.
Working Calibration Standards: Individual stock solutions containing 1mg/mL of each drug were prepared in methanol and stored at -20°C. Calibration curves were constructed from combined working standards prepared in MilliQ water at concentrations from 0.01 mcg/mL to 0.1 mcg/mL and 0.1mcg/mL to 10 mcg/mL . Calibration solutions were stored protected from light at 10°C for up to 4 hours prior to injection. 5µL injected. Chromatographic Conditions: LC Scouting & Optimization
Working from the LC conditions reported in Ref. 2, buffers at pH 8 and pH 10 were selected as starting points. The ACQUITY BEH C18 phase was selected based upon comparisons to the original analytical column, a Kromasil 100 C18 , 5 µm, 150 x 4 mm I.D. made in the Waters Column Selectivity Chart. http://www.waters.com A matrix (Figure 3) was generated using the following method scouting conditions: ACQUITY® TQD, ACQUITY FLR Detector, ACQUITY PDA, Waters 2777c High-Pressure Sample Manager or ACQUITY Sample Manger, ACQUITY Column Manger, ACQUITY Binary Solvent Manager. Split flow configuration to TQ and PDA>FLR. Sample: 50 mcg/mL mixture in 50%methanol 50%water. Column: 2.1 x 50mm ACQUITY UPLC® BEH C18 1.7μm Column Temp: 40°C Flow Rate: 0.7 mL/min Mobile Phase: A1: 10mM Ammonium Bicarbonate pH 10 A2: 10mM Ammonium Acetate pH 8.0 (pH 3 buffer is 2nd option in the normal scout protocol) B1: Acetonitrile (Optima) B2: Methanol (Optima) Gradient: 1—95% Organic in 10 minutes Inj Volume: 3μL of 50 mcg/mL mixed harmanes & DMT in water (10 μL loop) Weak Wash: 50/50 Water/Acetonitrile Strong Wash: 100% methanol 1% ammonium hydroxide ACQUITY PDA λ 210 to 500nm Rs: 1.2nm Date rate: 20pts. TC: Fast.
Optimization of detection conditions for TQ:
Figure 5: Top: IntelliStart™ automatically developed optimal TQD settings for analysis. Reference materials in 50% metha-nol & water under combined flow from reservoir and mobile phase were used to establish MRMs for each compound. UP to 4 Masses may be tuned for simultaneously. Bottom: Final MRM and MS1 Scan conditions generated by IntelliStart™
Compound Function Ion Mode
Start (min)
Stop (min) MRM
Dwell (sec)
Cone (V) CE (V)
Harmalol MRM ES+ 1.50 4.00 200.1 > 172.0 0.075 60 20
Harmaline MRM ES+ 3.00 6.00 215.1>174.1 0.100 30 24
Harmane MRM ES+ 3.00 6.00 183.1>142.1 0.100 45 26
Harmine MRM ES+ 3.00 6.00 213.1>169.9 0.100 25 34 Norhar‐mane MRM ES+ 3.00 6.00 169.1>115.1 0.100 60 32
DMT MRM ES+ 3.00 6.00 189.1 > 58.0 0.100 25 12
All MS1 SCAN ES+ 1.50 6.00
Mass Range: 70 to 230 Da; Scan Rate: 5000 Da/
sec 30
Source: 150° C; Desolvation: 350°C ; Desolvation gas: 600 L/Hr; Cone: 1 L/Hr.
Figure 3: PDA 238nm. 10 min gradients 1 to 95% were evaluated with both buffers and both organic solvents. The combination of pH 10 buffer & methanol (bottom plot) gave the best separation. At this point, modeling software Dry-Lab (http://www.molnar-institut.com/cd/e/dlw/drylab1.html) was used to optimize the separation. Final separation shown in Figure 4.
1
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% M
ETH
AN
OL
14.00
28.00
42.00
56.00
70.00
84.00
Minutes1.40 2.80 4.20 5.60 7.00
Figure 6: ACQUITY FLR 3D stop flow scan function en-ables accurate determination of optimal ex & em λ. 3D scan for harmine is shown.
Figure 4: Standard material. PDA Max Plot (240.0 to 350.0nm) and MRM 189.1>58 with % methanol overlay. Final Method Column:2.1 x 100mm ACQUITY UPLC® BEH C18 1.7μm; Column Temp:55°C; Flow Rate:0.5 mL/min. A1: 10mM Ammonium Bicarbonate pH 10; B2: Methanol Gradient:14% - 44% B2 0 to 2.50 min, hold 2min, 44% to 85% B at 6.50 min. Hold 1min, return to 14% B.
720002988en
