multi-channeling lc-ms/ms forensic methods for high...
TRANSCRIPT
Poster Note 64370
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol UseJoseph Di Bussolo,1 Ian White,2 Raidiri Castillo3 and Hashim Othman3 1Thermo Fisher Scientific, West Chester, PA; 2West Chester University of Pennsylvania, West Chester, PA; 3BioReference Laboratories, Elmwood Park, NJ
2Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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Modified Prelude
EtS
For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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Modified Prelude
EtS
For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
3Thermo Scientific Poster Note • MASCL • PN64370-EN 0315S
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
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For forensic toxicology use only.
Conclusions The modified Prelude - Endura system, which utilized unique positive-
displacement pumps, produced results for Bup/Norbup and EtG/EtS methods that were consistent with those produced by the conventional Transcend - Endura system.
The maximum throughput from both 4-channel systems was 34 injections per hour when multiplexing the two forensic methods across all four channels.
The modified Prelude system reduced solvent consumption by at least 60%. Other benefits of using this system were:
Avoided pulsations of reciprocating pumps
Easier to use, purge, prepare and maintain
Smaller footprint compared to Transcend LX4
Overview Purpose: Compare the performance of running LC-MS/MS forensic methods used to detect buprenorphine and ethanol use on two different four-channel UHPLC systems - one utilizing conventional reciprocating pumps and the other using positive-displacement pumps.
Methods: Reversed-phase liquid chromatography of analytes with corresponding stable-isotope internal standards eluting from up to four UHPLC channels into a heated electro-spray ionization (HESI) source of a triple- quadrupole mass spectrometer were used to measure urine levels of the forensic compounds Buprenorphine (Bup) & Norbuprenorphine (Norbup) after hydrolysis and Ethyl-Glucuronide (EtG) and Ethyl Sulfate (EtS) after dilution.
Results: Running Bup/Norbup batches on two channels while EtG/EtS batches ran on the remaining two channels yielded a throughput of 17 injections per hour for each batch, which totaled 34 injections per hour. The four-channel system with positive-displacement pumps reduced solvent consumption by at least 60%. Results from this system were within +/- 15% of those determined on a conventional multichannel system using reciprocating pumps.
Introduction Many forensic laboratories run several different LC-MS methods in series on a single-channel LC-MS system. If the methods involve different ion sources, columns and mobile phases, the changeover is time consuming, labor intensive and increases the risk of mistakes and contamination. A four-channel UHPLC system multiplexed into one mass spectrometer permits parallel batches of up to four different methods utilizing a common ion source and unique columns and mobile phases to be completed in a fraction of the time and effort. However, conventional systems utilizing reciprocating pumps needlessly consume mobile-phase solvents between injections. The Thermo Scientific™ Prelude™ SPLC system, which utilizes positive-displacement pumps does not waste solvents between injections. A modified version of this multi-channel UHPLC system was tested by running batches for two forensic methods - Bup/Norbup and EtG/EtS - and comparing those results to those from a conventional UHPLC system that ran the same sets of calibrators, QCs and specimens.
Mass Spectrometry System Control & Data Analysis
Thermo Scientific™ TraceFinder™ with Aria™ MX software was used to control the Transcend II LX4 and Thermo Scientific™ TSQ Endura™ MS/MS systems, submit batches to desired channels as well as for analyzing data and reporting results.
The Endura triple-quadrupole mass spectrometer was used with HESI. Ion source and MS/MS conditions are described in Figure 4.
FIGURE 4. Mass spectrometry conditions.
Common source
conditions:
Bup/Norbup
acquisition
method:
EtG/EtS
acquisition
method:
FIGURE 5. Multi-channeling Bup/Norbup and EtG/EtS batches.
FIGURE 5. Calibration Plots from Modified Prelude - Endura system.
Multi-channeling LC-MS/MS Forensic Methods for High-Throughput Urine Screening to Detect Buprenorphine and Ethanol Use Joseph Di Bussolo1, Catherine Lafontaine1, Mercedes Castillo2, Thomaskutty Thomas2 & Hashim Othman2. 1Thermo Fisher Scientific, West Chester, PA, 2BioReference Laboratories, Elmwood Park, NJ
Methods Consumables
HPLC-grade solvents, reagents and most other consumables were from Thermo Fisher Scientific. Buprenorphine and norbuprenorphine and their corresponding deuterated internal standards as well as ethyl sulfate and ethyl glucuronide and their corresponding -deuterated internal standards were from Cerilliant (Round Rock, TX). Calibrators were made by mixing these standards with synthetic urine. Liquid urine controls were from Biochemical Diagnostics (Edgewood, NY). β-glucuronidase powder was purchased from Sigma-Aldrich.
Sample Preparation:
Urine specimens and corresponding calibrators and QCs to be analyzed for Bup/Norbup were hydrolyzed by incubating a mixture of 150 µL of β-glucuronidase solution (10,000 U/mL, pH 5) with 200 µL of specimen, and 50 µL of IS solution containing - buprenorphine-D3 & norbuprenorphine-D4 for 1.5 hours at 60°C. Each preparation was then mixed with 200 µL of cold methanol and refrigerated for 10 minutes before centrifugation. 20 µL injections of supernatants from each preparation were made into the UHPLC systems.
Urine specimens and corresponding calibrators and QCs to be analyzed for EtG/EtS were diluted 1:10 with water and then spiked with 50 µL of IS solution containing EtG-D5 & EtS-D5 before making 20 µL injections into the UHPLC system.
Liquid Chromatography
The “conventional” multi-channel LC system was a Thermo Scientific™ Transcend™ LX4 equipped with binary-solvent pumps and a dual-arm autosampler configuration. Thermo Scientific™ Accucore™ RP-MS, 2.6 µm, 50 x 2.1 mm HPLC columns were used for the Bup/Norbup method. Mobile phase conditions for this method are described in Figure 1. Thermo Scientific™ Synchronis™ aQ, 5 µm, 50 x 3.0 mm HPLC columns were used for the EtG/EtS method. Mobile phase conditions for this method are described in Figure 2.
A Thermo Scientific™ Prelude™ SPLC system was modified to permit injections across four channels, similar to the Transcend LX4 system. The Bup/Norbup method on this system utilized the same column and mobile phase conditions as the Transcend system. However, the EtG/EtS method was modified to use the Thermo Scientific™ Synchronis™ aQ, 3 µm, 100 x 3.0 mm HPLC columns, as shown in Figure 3.
Results Throughput and Multi-channeling Bup/Norbup and EtG/EtS batches
Multi-channeling Bup/Norbup across 2 channels while EtG/EtS runs on one allowed 23 Bup/Norbup and 12 EtG/EtS injections/hour. Using 2 channels for each does not increase throughputs but ensures completion of all batches even if one channel stops because of leakage or over-pressurization. Multi-channeling two batches is illustrated in Figure 5.
FIGURE 1. Transcend Bup/norbup LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
Comparison between Transcend and Modified Prelude Systems
Batches of calibrators, QCs and at least 20 specimens were prepared and shared between the two multi-channel systems. Typical calibration plots from the modified Prelude - Endura system are shown in Figure 5. Desired quantitation ranges were consistently linear (r2 > 0.99 with 1/X weighting) whether when calibrators were injected into one channel or across all channels.
Only results that were within the analytical ranges for each method were used for comparisons. Differences in calculated amounts averaged less than +/- 5% within a maximum of +/- 15%. Scatter plots in Figure 6 showed good correlations between the two systems.
FIGURE 6. Scatter plots of results - Transcend vs. modified Prelude.
FIGURE 2. Transcend EtG/EtS LC conditions.
Start data 0.5 min Data window: 1.5 min Total run time: 4.0 min
FIGURE 3. Modified Prelude EtG/EtS LC conditions.
Start data 0.75 min Data window: 1.5 min Total run time: 4.0 min
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For forensic toxicology use only.
©2015 Thermo Fisher Scientific. All Rights Reserved.
Cerilliant is a registered trademark of the Sigma-Aldrich Corporation. Biochemical Diagnostics is a registered trademark of Biochemical Diagnostics, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.