Upload File

isolation of drug metabolites from urine using tandem

  • Home
  • Documents
  • Isolation of Drug Metabolites from Urine using Tandem
1
©2005 Waters Corporation TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS Isolation of Drug Metabolites from Urine using Tandem Quadrupole (MS/MS) Directed Purification Paul Lefebvre*, Robert S. Plumb, Ronan Cleary, and Warren B. Potts III Waters Corporation, 34 Maple Street, Milford MA 01757 MRM Collection With MRM (Multiple Reaction Monitoring) data acquisition, MS1 is static on the parent mass and MS2 is static on a specific daughter ion. By selectively detecting a product ion, the signal-to-noise ratio is optimized, thus reducing the isobaric interference and allow only the target to be collected. This mode of acquisition requires previous knowledge of the exact parent and the exact daughter ions before purification. This example data shows the MRM acquisition and collection of the caffeine metabolites. The metabolites of interest for isolation have the transitions 181 to 134 and 167 to 110 Fractionation based on MRM acquisition • For the peak present in the chromatogram, both the specific parent and the specific daughter ion. • The chromatogram shows that only one fraction for each mass was collected. Note: Only 1 of the 2 possible dimethyl metabolites was present in this sample. Further analysis is necessary to determine its identity. References Methods The identification of drug metabolites following animal or human volunteer stud- ies is essential to the drug discovery & development process and regulatory sub- missions. Traditionally this has been achieved by the use of Liquid chromatog- raphy or gas chromatography coupled to mass spectrometry [1-2]. More re- cently the use of hyphenated techniques such as LC/NMR and LC/NMR/MS have become more common place in the drug metabolism laboratory allowing a more precise identification of the site of metabolism [3-4]. While LC/NMR and LC/NMR/MS are extremely powerful tools they are low throughput and sensitivity limited. The loading capacity of analytical columns restricts the amount of material that can be loaded on to the column before the column ex- hibits either volume or mass overloading effects and the chromatographic reso- lution is lost. Thus making it less attractive for the analysis of highly potent com- pounds dosed at low levels or those compound that undergo extensive metabo- lism. In such cases it is often necessary to perform a pre-concentration step such as SPE or L/L extraction, these are both time consuming and risk loosing valuable information. The use of MS directed auto-purification, using semi preparative scale columns (typically 19mm i.d), is now common place within the pharmaceutical industry, especially to support lead candidate purification. This approach has also been applied to the isolation of drug metabolites with some success [5]. The extra sensitivity and selectivity of MS/MS mass spectrometry should allow for the more precise selection of drug metabolites, and the use of neutral loss and pre- cursor ion scanning detection modes will facilitate the collection of drug metabo- lites without the need for prior knowledge of compound metabolism. This paper shows how tandem quadrupole mass spectrometry has been em- ployed with both analytical and semi-preparative scale chromatography for the isolation of the metabolites of common pharmaceuticals, from urine. The appli- cation of MRM, neutral loss and precursor ion scanning is demonstrated. We also demonstrate how the use of MS/MS directed purification facilitates the combination of samples from several chromatographic runs Overview Ibuprofen Metabolites Separation Water: Acetonitrile: 10 mM ammonium for- mate, 1.25 ml/min total flow gradient. 0 – 5 minutes: 5%, 5 – 35 minutes: 5 – 60% B, 35 – 35.5 minutes: 60 – 95% B, 35.5 – 39.5 min- utes: 95% B, 39.5 – 40 minutes: 95 – 5% B, 45 minutes end MS detection Electrospray negative, 3 kV capillary voltage, 30 V cone voltage, 20 V collision energy Ibuprofen gluceronide metabolite with a com- mon daughter ion on 193 7 Caffeine Metabolites Separation Water: Acetonitrile: 0.1%Formic acid, 1.25 ml/min total flow gradient. 0 – 5 minutes: 0%, 5 – 35 minutes: 0 – 10% B, 35 – 35.5 minutes: 10 – 95% B, 35.5 – 39.5 minutes: 95% B, 39.5 – 40 minutes: 95 – 5% B, 45 minutes end MS detection Electrospray positive, 3 kV capillary voltage, 30 V cone voltage, 20 V collision energy (for MS/MS experiments) Metabolism of caffeine by demethylation. Metabolites which maintain the methyl group in the 1 position have a common fragment of 57. Paraxanthine – m/ z = 181 1,7 Dimethylxanthine Caffeine - m/ z =195 1,3,7 Trimethylxanthine Theophylline – m/ z = 181 1,3 Dimethylxanthine 1Methylxanthine – m/ z = 167 Common fragment of xanthine a methyl in position 1 - m/ z = 57 With single quadrupole directed purification, all ions generated in the source are passed through the quadrupole and detected. This mode is possible on the Quattro Micro by using the “Scan” mode of acquisition. Only MS1 is scanned and there is no collision energy or scanning of Q3. Because all the ions generated are detected, complex mixtures can contain numerous isobaric interferences. Consequently, multiple fractions can be generated from a single m/z value. Additional analysis is then required to identify fraction of interest. Example Data - Caffeine metabolites m/z = 167 and 181 • 8 fractions collected for m/z 167 and 5 fractions collected for m/z 181 • Additional analysis required to determine fraction of interest Fractionation based only on scanning the 1st quadrupole MS1 static MS2 static Collision Cell RF only (all masses pass) Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 M/ Z = 167 M/ Z = 181 OR Constant Neutral Loss Collection With constant neutral loss acquisition, MS1 and MS2 are scanned in synchronization. When MS1 transmits a specific parent ion, MS2 “looks” for a daughter which is the parent minus the neutral loss value. If the correct daughter is present, it registers at the detector. The constant neutral loss “spectrum” shows the only the masses of all the parents that lost the specific mass. MS1 scanning MS2 scanning Collision Cell RF only (all masses pass) Constant Neutral Loss Example Data This example data shows the constant neutral loss of 57 acquisition and collection of the caf- feine metabolites with m/z = 167 and 181. Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 % 0 m/z 100 120 140 160 180 200 % 0 100 166.9 114.4 m /z 100 120 140 160 180 200 % 0 100 181.0 182.0 M/ Z = 181 M/ Z = 167 N eutral loss of 57 TIC • 2 Fractions are collected, 1 for each mass. • Fractions contain the target mass and have the specific neutral loss Fractionation based on constant neutral loss acquisition Applications for Fraction Collection from Constant Neutral Loss Acquisition Mass Triggered Collection With constant neutral loss acquisition, the only peaks detected are the ones with the loss of the specific mass, in this case 57. Depending on the specificity of the loss, numerous ions can be detected. This leads to complex total ion chromatograms. Therefore, when triggering by a specific mass, the collected target must contain the parent of interest and have a specific neutral loss. Collection Triggered on TIC When using this mode of acquisition and collection, all the peaks with a specific neutral loss are collected. This is valuable when the metabolites have a specific loss related to the drug’s structure. It could also be used for isolating a class of metabolites with a generic loss, for example sulfates (- 80) or glucuronides (- 176). The parent mass for each fraction can then be extracted and used to aid in the identification of the metabolites. In the constant neutral loss example shown, collection could have also been triggered from the TIC. All peaks in the –57 TIC would be collected and additional analysis or data review would be required to find the desired fractions. Precursor Ion Collection With precursor ion acquisition, MS1 is scanning and MS2 is fixed on a specific daughter ion. If the specific daughter ion is observed, it is registered and the detector. The “spectrum” only shows the masses which have that specific daughter. Fraction collection from a precursor ion acquisition has to be from the TIC, since the parent mass is unknown. This mode of fraction collection is valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected. MS1 scanning MS2 static Collision Cell RF only (all masses pass) Precursor Ion Example Data This example data shows the collection of ibuprofen metabolites from the precursor ion of 193 acquisition. • 3 Fractions are collected, M/Z = 273, 397, and 381 • 2 of the 3 fractions were previous identified by Kearney et al., the 3rd is an unknown and requires additional analysis to determine structure and if it is a metabolite of the ibuprofen • The ESCi Multi-Mode ionization source allows for both ESI +/- and APCI +/- acquisition to occur within the same run. This allows for fraction collection to be triggered form any of the acquisition channels, thus proving useful if the metabolites require different ionization. With these type of samples, the only options for collection would be to split the sample and run in different modes or time base fractionate and the analysed all the fractions by both modes to determine the targets. • Mixed trigger fractionation allows for boolean logic strings to trigger collection from multiple data traces. For example, collection can occur only when Mass A is present and Mass B is not or when a peak is present in 2 different traces. • Fraction collection with a tandem quadrupole mass spectrometer is now possible using 4 different modes of data acquisition, scan, MRM, constant neutral loss and precursor ion, allowing for improved versatility for fraction collection options. • MRM is the most selective by only monitoring a specific parent and a specific daughter. This greatly reduces the isobaric interferences but requires previous knowledge of the parent / daughter transition. • Constant neutral loss can be used for a collecting a class of compounds with a target specific loss or a mode generic loss for a broader study. It can also be used as a second filter, where the target has to have a specific mass and the neutral loss. • Collection from a precursor ion TIC scan allows for all the parents with a specific daughter ion to be collected. This mode of collection can be valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected. • The different modes of collection have different applications based on 1) the requirements of the study, 2) the level of previous knowledge about the sample and 3) the characteristics of the sample like ionization and fragmentation. Conclusions 1. Ismail IM. Dear GJ.. Xenobiotica. 29(9):957-967, 1999 2. Dear GJ, Mallett DN and Plumb RS. LC-GC Europe 14(10) 616-624, 2001 3. Dear GJ, Plumb RS, Sweatman BC, Parry PS, Robert AD, Lindon JC, Nicholson JK and Ismail IM. Journal of Chromatogra- phy B 748 295-309 2000 4. G. J Dear, R. S. Plumb, B. C. Sweatman, J. Ayrton, J. C. Lindon, J. K. Nicholson, , J. Chromatogr. B, 748, 281-293 2000 5. Plumb RS. Ayrton J. Dear GJ. Sweatman BC. Ismail IM.. Rapid Communications in Mass Spectrometry. 13(10):845-854, 1999 6. Bendriss E, Markoglou N, Wainer IW. Journal of Chromatography B 746 331-338 2000 7. Kearney et al. “Exact Mass MS/MS of Ibuprofen Metabolites using Hybrid Quadrupole-Orthogonal Time-of-Flight Mass Spectrometry Equipped with a Lockspray Source”, Waters Corporation Application Note # 720000706EN Additional Collection Options Fractionation based on the precursor ions of 193 TIC acquisition. Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 7 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 Time 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 7 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 % 0 m /z 200 300 400 500 600 700 800 % 0 100 273.1 274.7 m /z 200 300 400 500 600 % 0 100 397.0 273.1 397.8 398.7 m /z 200 300 400 500 600 % 0 100 381.2 382.0 383.4 Parents of 193 TIC M/Z 381 M/Z 397 M/Z 273 Tandem Quadrupole Purification Single Quadrupole Purification Lit Code Number 720001199EN

Upload: others

Post on 05-Nov-2021

8 views

Category:

Documents


0 download

Report

TRANSCRIPT

Page 1: Isolation of Drug Metabolites from Urine using Tandem

©2005 Waters Corporation

TO DOWNLOAD A COPY OF THIS POSTER VISIT WWW.WATERS.COM/POSTERS

Isolation of Drug Metabolites from Urine using Tandem Quadrupole (MS/MS) Directed Purification

Paul Lefebvre*, Robert S. Plumb, Ronan Cleary, and Warren B. Potts III Waters Corporation, 34 Maple Street, Milford MA 01757

MRM Collection With MRM (Multiple Reaction Monitoring) data acquisition, MS1 is static on the parent mass and MS2 is static on a specific daughter ion. By selectively detecting a product ion, the signal-to-noise ratio is optimized, thus reducing the isobaric interference and allow only the target to be collected. This mode of acquisition requires previous knowledge of the exact parent and the exact daughter ions before purification.

This example data shows the MRM acquisition and collection of the caffeine metabolites. The metabolites of interest for isolation have the transitions 181 to 134 and 167 to 110

Fractionation based on MRM acquisition

• For the peak present in the chromatogram, both the specific parent and the specific daughter ion.

• The chromatogram shows that only one fraction for each mass was collected. Note: Only 1 of the 2 possible dimethyl metabolites was present in this sample. Further analysis is necessary to determine its identity.

References

Methods

The identification of drug metabolites following animal or human volunteer stud-ies is essential to the drug discovery & development process and regulatory sub-missions. Traditionally this has been achieved by the use of Liquid chromatog-raphy or gas chromatography coupled to mass spectrometry [1-2]. More re-cently the use of hyphenated techniques such as LC/NMR and LC/NMR/MS have become more common place in the drug metabolism laboratory allowing a more precise identification of the site of metabolism [3-4]. While LC/NMR and LC/NMR/MS are extremely powerful tools they are low throughput and sensitivity limited. The loading capacity of analytical columns restricts the amount of material that can be loaded on to the column before the column ex-hibits either volume or mass overloading effects and the chromatographic reso-lution is lost. Thus making it less attractive for the analysis of highly potent com-pounds dosed at low levels or those compound that undergo extensive metabo-lism. In such cases it is often necessary to perform a pre-concentration step such as SPE or L/L extraction, these are both time consuming and risk loosing valuable information. The use of MS directed auto-purification, using semi preparative scale columns (typically 19mm i.d), is now common place within the pharmaceutical industry, especially to support lead candidate purification. This approach has also been applied to the isolation of drug metabolites with some success [5]. The extra sensitivity and selectivity of MS/MS mass spectrometry should allow for the more precise selection of drug metabolites, and the use of neutral loss and pre-cursor ion scanning detection modes will facilitate the collection of drug metabo-lites without the need for prior knowledge of compound metabolism. This paper shows how tandem quadrupole mass spectrometry has been em-ployed with both analytical and semi-preparative scale chromatography for the isolation of the metabolites of common pharmaceuticals, from urine. The appli-cation of MRM, neutral loss and precursor ion scanning is demonstrated. We also demonstrate how the use of MS/MS directed purification facilitates the combination of samples from several chromatographic runs

Overview

Ibuprofen Metabolites Separation Water: Acetonitrile: 10 mM ammonium for-mate, 1.25 ml/min total flow gradient. 0 – 5 minutes: 5%, 5 – 35 minutes: 5 – 60% B, 35 – 35.5 minutes: 60 – 95% B, 35.5 – 39.5 min-utes: 95% B, 39.5 – 40 minutes: 95 – 5% B, 45 minutes end MS detection Electrospray negative, 3 kV capillary voltage, 30 V cone voltage, 20 V collision energy Ibuprofen gluceronide metabolite with a com-

mon daughter ion on 1937

Caffeine Metabolites

Separation Water: Acetonitrile: 0.1%Formic acid, 1.25 ml/min total flow gradient. 0 – 5 minutes: 0%, 5 – 35 minutes: 0 – 10% B, 35 – 35.5 minutes: 10 – 95% B, 35.5 – 39.5 minutes: 95% B, 39.5 – 40 minutes: 95 – 5% B, 45 minutes end MS detection Electrospray positive, 3 kV capillary voltage, 30 V cone voltage, 20 V collision energy (for MS/MS experiments)

Metabolism of caffeine by demethylation. Metabolites which maintain the methyl group in the 1 position have a common fragment of 57.

Paraxanthine – m/ z = 181 1,7 Dimethylxanthine

Caffeine - m/ z =1951,3,7 Trimethylxanthine

Theophylline – m/ z = 1811,3 Dimethylxanthine

1Methylxanthine – m/ z = 167

Common fragment of xanthine a methyl in

position 1 - m/ z = 57

With single quadrupole directed purification, all ions generated in the source are passed through the quadrupole and detected. This mode is possible on the Quattro Micro by using the “Scan” mode of acquisition. Only MS1 is scanned and there is no collision energy or scanning of Q3. Because all the ions generated are detected, complex mixtures can contain numerous isobaric interferences. Consequently, multiple fractions can be generated from a single m/z value. Additional analysis is then required to identify fraction of interest. Example Data - Caffeine metabolites m/z = 167 and 181

• 8 fractions collected for m/z 167 and 5 fractions collected for m/z 181

• Additional analysis required to determine fraction of interest

Fractionation based only on scanning the 1st quadrupole

MS1static

MS2static

Collision CellRF only

(all masses pass)

MS1static

MS2static

Collision CellRF only

(all masses pass)

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00

%

0

M / Z = 167

M / Z = 181

O R

Constant Neutral Loss Collection With constant neutral loss acquisition, MS1 and MS2 are scanned in synchronization. When MS1 transmits a specific parent ion, MS2 “looks” for a daughter which is the parent minus the neutral loss value. If the correct daughter is present, it registers at the detector. The constant neutral loss “spectrum” shows the only the masses of all the parents that lost the specific mass.

MS1scanning

MS2scanning

Collision CellRF only

(all masses pass)

MS1scanning

MS2scanning

Collision CellRF only

(all masses pass)

Constant Neutral Loss Example Data This example data shows the constant neutral loss of 57 acquisition and collection of the caf-feine metabolites with m/z = 167 and 181.

T im e2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

T im e2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

2 .5 0 5 .0 0 7 .50 1 0 .00 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 25 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0 4 5 .0 0

%

0

m /z1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0

%

0

1 00 16 6 .9

1 1 4 .4

m /z1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0

%

0

1 0 0 1 8 1 .0

1 8 2 .0

M / Z = 1 8 1

M / Z = 1 6 7

N eutra l lo ss o f 5 7 TIC

• 2 Fractions are collected, 1 for each mass. • Fractions contain the target mass and have the specific neutral loss

Fractionation based on constant neutral loss acquisition

Applications for Fraction Collection from Constant Neutral Loss Acquisition Mass Triggered Collection

With constant neutral loss acquisition, the only peaks detected are the ones with the loss of the specific mass, in this case 57. Depending on the specificity of the loss, numerous ions can be detected. This leads to complex total ion chromatograms. Therefore, when triggering by a specific mass, the collected target must contain the parent of interest and have a specific neutral loss. Collection Triggered on TIC When using this mode of acquisition and collection, all the peaks with a specific neutral loss are collected. This is valuable when the metabolites have a specific loss related to the drug’s structure. It could also be used for isolating a class of metabolites with a generic loss, for example sulfates (- 80) or glucuronides (- 176). The parent mass for each fraction can then be extracted and used to aid in the identification of the metabolites. In the constant neutral loss example shown, collection could have also been triggered from the TIC. All peaks in the –57 TIC would be collected and additional analysis or data review would be required to find the desired fractions.

Precursor Ion Collection With precursor ion acquisition, MS1 is scanning and MS2 is fixed on a specific daughter ion. If the specific daughter ion is observed, it is registered and the detector. The “spectrum” only shows the masses which have that specific daughter. Fraction collection from a precursor ion acquisition has to be from the TIC, since the parent mass is unknown. This mode of fraction collection is valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected.

MS1scanning

MS2static

Collision CellRF only

(all masses pass)

MS1scanning

MS2static

Collision CellRF only

(all masses pass)

Precursor Ion Example Data This example data shows the collection of ibuprofen metabolites from the precursor ion of 193 acquisition.

• 3 Fractions are collected, M/Z = 273, 397, and 381 • 2 of the 3 fractions were previous identified by Kearney et al., the 3rd is an unknown and

requires additional analysis to determine structure and if it is a metabolite of the ibuprofen

• The ESCi Multi-Mode ionization source allows for both ESI +/- and APCI +/- acquisition to occur within the same run. This allows for fraction collection to be triggered form any of the acquisition channels, thus proving useful if the metabolites require different ionization. With these type of samples, the only options for collection would be to split the sample and run in different modes or time base fractionate and the analysed all the fractions by both modes to determine the targets.

• Mixed trigger fractionation allows for boolean logic strings to trigger collection from multiple data traces. For example, collection can occur only when Mass A is present and Mass B is not or when a peak is present in 2 different traces.

• Fraction collection with a tandem quadrupole mass spectrometer is now possible using 4 different modes of data acquisition, scan, MRM, constant neutral loss and precursor ion, allowing for improved versatility for fraction collection options.

• MRM is the most selective by only monitoring a specific parent and a specific daughter. This greatly reduces the isobaric interferences but requires previous knowledge of the parent / daughter transition.

• Constant neutral loss can be used for a collecting a class of compounds with a target specific loss or a mode generic loss for a broader study. It can also be used as a second filter, where the target has to have a specific mass and the neutral loss.

• Collection from a precursor ion TIC scan allows for all the parents with a specific daughter ion to be collected. This mode of collection can be valuable when the metabolites are unknown, but there is a common fragment of the core compound that can be detected.

• The different modes of collection have different applications based on 1) the requirements of the study, 2) the level of previous knowledge about the sample and 3) the characteristics of the sample like ionization and fragmentation.

Conclusions

1. Ismail IM. Dear GJ.. Xenobiotica. 29(9):957-967, 1999

2. Dear GJ, Mallett DN and Plumb RS. LC-GC Europe 14(10) 616-624, 2001

3. Dear GJ, Plumb RS, Sweatman BC, Parry PS, Robert AD, Lindon JC, Nicholson JK and Ismail IM. Journal of Chromatogra-phy B 748 295-309 2000

4. G. J Dear, R. S. Plumb, B. C. Sweatman, J. Ayrton, J. C. Lindon, J. K. Nicholson, , J. Chromatogr. B, 748, 281-293 2000

5. Plumb RS. Ayrton J. Dear GJ. Sweatman BC. Ismail IM.. Rapid Communications in Mass Spectrometry. 13(10):845-854, 1999

6. Bendriss E, Markoglou N, Wainer IW. Journal of Chromatography B 746 331-338 2000 7. Kearney et al. “Exact Mass MS/MS of Ibuprofen Metabolites using Hybrid Quadrupole-Orthogonal Time-of-Flight Mass

Spectrometry Equipped with a Lockspray Source”, Waters Corporation Application Note # 720000706EN

Additional Collection Options

Fractionation based on the precursor ions of 193 TIC acquisition.

T im e2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

7

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

T im e2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

7

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

2 .5 0 5 .0 0 7 .5 0 1 0 .0 0 1 2 .5 0 1 5 .0 0 1 7 .5 0 2 0 .0 0 2 2 .5 0 2 5 .0 0 2 7 .5 0 3 0 .0 0 3 2 .5 0 3 5 .0 0 3 7 .5 0 4 0 .0 0 4 2 .5 0

%

0

m /z2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0

%

0

1 0 0 2 7 3 .1

2 7 4 .7

m /z2 0 0 3 0 0 4 0 0 5 0 0 6 0 0

%

0

1 0 0 3 9 7 .0

2 7 3 .1

3 9 7 .8

3 9 8 .7

m /z2 0 0 3 0 0 4 0 0 5 0 0 6 0 0

%

0

1 0 0 3 8 1 .2

3 8 2 .0

3 8 3 .4

Pa ren ts o f 1 9 3 TIC

M / Z 3 8 1

M / Z 3 9 7

M / Z 2 7 3

Tandem Quadrupole Purification

Single Quadrupole Purification

Lit Code Number 720001199EN

Analysis of Uric Acid Metabolites in Urine (or in ...collection.asdlib.org/wp-content/uploads/2017/02/Urine_Report2.pdf · Analysis of Uric Acid Metabolites in Urine (or in synthetic

Hormone Metabolites in Dried Urine - ZRT Laboratory · 2018-09-26 · 866.600.1636 infortlab.com rtlab.com PAGE 1 Hormone Metabolites in Dried Urine Adrenal Profile: A picture of

Secondary metabolites

static-content.springer.com10.1007/s113…  · Web viewSeparate word file with all annotated drug related metabolites. ... 4 unique urine extracts contained one or more metformin

A NEW STRATEGY FOR THE DETERMINATION OF CAPTAN … · chromatography-tandem mass spectrometry (LC/APCI-MS/MS) method for the quantification of captan and folpet phthalimide metabolites

Determination of Multi-Residue Tetracyclines and … of Multi-Residue Tetracyclines and their Metabolites in Milk by High Performance Liquid Chromatography - Tandem Mass Spectrometry

THE ANALYSIS OF CANNABlNOlDS IN BlOlOGlCAL FLUIDS · Urine v Anthony C. Moffat, Peter L. Williams, and Laurence J. King An EMIT Assay for Cannabinoid Metabolites in Urine M. J. deLaurentis,

Primary metabolites

Phthalate Metabolites Urine HPLC/ESI-MS/MS 6306.03 July 3 ...Phthalate Metabolites in Urine NHANES 2007-2008 ... entered into the Access database before sample preparation. If possible,

Comprehensive LC/MS Analysis of Opiates, Opioids ... · Comprehensive LC/MS Analysis of Opiates, Opioids, Benzodiazepines, Amphetamines, Illicits, and Metabolites in Urine Application

Petroleum Metabolites

Determination of Amphetamine and Derivatives in … · Determination of Amphetamine and Derivatives in Urine Using a Modified QuEChERS and Capillary Electrophoresis Tandem Mass Spectrometry

Urine Examination (Urine Analysis or Urine Report)

Trends in Analytical Chemistry, Vol. 26, No. 2, 2007 ... · Recent trends in liquid chromatography-tandem mass spectrometry to determine pesticides and their metabolites ... methods

New cathinone-derived designer drugs 3-bromomethcathinone ... · FMC phase I and phase II metabolites in rat urine and human liver microsomes using gas chromatography-mass spectrometry

Analysis and identification of abnormal metabolites in urine using : Chromatography

Seconadry metabolites

Identification of 2-[2-nitro-4-(trifluoromethyl)benzoyl ... · Regular paper Identification of 2-[2-nitro-4-(trifluoromethyl)benzoyl]- cyclohexane-1,3-dione metabolites in urine of

Secondary Metabolites from Plants Plant Secondary Metabolites

GLYPHOSATE (158) AND METABOLITES … · GLYPHOSATE (158) AND METABOLITES ... urine and faeces samples were taken prior to the first dose and at 24 h ... and by further extraction

Identification of metabolites: analytical challenges for ... · Metabolite characterisation: Mass spectrometry Tandem Mass Spectrometry technique to identify metabolites in complex

Analysis of polar urinary metabolites for metabolic phenotyping … · 2017-02-10 · the separation of a mixture of polar metabolites in human urine using an optimised 7min gradient

Poisoning with Soman, an Organophosphorus … with Soman, an Organophosphorus Nerve Agent, Alters Fecal Bacterial Biota and Urine Metabolites: a Case for Novel Signatures for Asymptomatic

Profiling Hydrocodone and - SAMHSA Hydrocodone and Metabolites in Urine ... Introduction to Study Design • Hydrocodone ... these data suggest that drug testing requirements for HC

Structure elucidation of ten autotaxin inhibitors and their … · and their metabolites using Liquid Chromatography tandem Mass Spectrometry ... MSc Chemistry Analytical Sciences

Review Structure elucidation of phase II metabolites by …€¦ ·  · 2017-10-24Structure elucidation of phase II metabolites by tandem mass spectrometry: ... Glucose 180c Cys–Gly

Heather M. Stapleton, Ph - Nicholas School Stapleton_062017.pdf · Heather M. Stapleton, Ph.D Duke University, Nicholas School of the Environment ... (TPHP) Metabolites in Human Urine

Anti metabolites

Lee A. Ellingson, CRNA, MHS - NDANA analgesia - Old New.pdfSulfate conjugation ... Metabolites excreted in urine. Morphine sparing effect: ... (Some studies use 300 mg) Tylenol 1 Gm

PRECISION INSTRUMENTS USER GUIDETandem Tandem Tandem Tandem Tandem Tandem Tandem INTERNATIONAL LIMITED WARRANTY

Organophosphate metabolites in urine samples from Danish ... · som bliver brugt som biomarkører for eksponering for organofosfat-insekticider som dichlorvos, fenthion, dimethoat,

Integration of Metabolism. FUELS, METABOLITES AND DISORDERS FUELS URINE BILE/FECES METABOLITES METABOLITES Starch, Glucose NH 4, + SO 4 2-, HPO 4 2- Cholesterol,

Testing Urine Hormone Metabolites

Famciclovir (FAMVIR(R))downloads.hindawi.com/journals/idog/1997/508483.pdfFAMCICLOVIR (FAMVIR(R)) vir.4,s Little or nofamcicloviris detected in plasma or urine. Inactive metabolites

How soon after intake can drug metabolites be …...Academiejaar 2010 - 2011 How soon after intake can drug metabolites be detected in urine? Karen MEERT Promotor: Prof. Dr. Alain