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Application of Structure-Based LC/MS Database Management for Forensic Analysis

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Waters Corporation, Milford, MA USA

Antony Williams, Vitaly Lashin and Ilya Troisky

Advanced Chemistry Development, Toronto, Ontario, Canada

4Chromatography

– Instrument: Waters 2690 Alliance™ System under MassLynx control

– Solvents: A: H2O, B: MeCN

§ 200 ml/min

§ 30-40% B in 20 min

§ 35 min total run time – 2.1x100 mm XTerraMS™ C18,

3.5 mm

§ 35 C column temp 4PDA

– Instrument: Waters Model 996 Photodiode Array Detector – 210-400 nm

4Samples

– Standards diluted in 30/70 MeCN/H2O

4Mass Spectrometry

– Instrument: Waters ZQ™ 4000 under MassLynx control – ESI+, centroid – Scan 100-600 amu – Scan time: 0.2 sec – Interscan delay: 0.1 sec – Tune page settings

§ Capillary voltage: 3.0 kV

§ Cone voltage: 30, 50, 70 V

§ Extractor: 6 V

§ RF lens: 0.1 V

§ Ion energy: 0.1

§ LM & HM resolution: 14.5

&15.0

§ Source temperature: 150 C

§ Desolvation temperature: 300 C

§ Cone gas: 150 l/hr

§ Desolvation gas: 600 l/hr

LC/UV/MS Conditions Overview

4 Application of LC/UV/MS to the analysis of toxicologically relevant samples

4 Demonstration of several ways in which structural information may assist in sample identification

– Use of a structure-based data management system for manual interpretation and analysis of sample components

– Software tools to interpret difficult analysis cases (e.g., high noise backgrounds, manual and automated fragment assignment tools)

Introduction

4 In many cases, an automated search method can be used for sample identification

– However...

§Automated search results still must be manually reviewed

§Difficult or low concentration samples require significant manual interpretation

§Commercially available databases are only available with nominal mass accuracy

4 Integration of structures with spectral information can provide improved data management and enhance library searching capabilities

– Particularly useful for manual interpretation and analysis of results – Provides structure and substructure search capabilities for data mining – Allows management of multiple forms of MS data with a single chemical

structure – EI, ESI, CI, etc.

30 mg Waters Oasis HLB Cartridge Condition 1 ml MeOH and 1 ml H2O

Load

1 ml spiked serum or Hanks’ solution (10:1 dilution of standards with serum or Hanks’)

Wash 1 ml 5% MeOH in H2O

Elute 1 ml MeOH Evaporate and Reconstitute Room temp in Savant Speed Vac 200 ml mobile phase (30% MeCN in H2O)

Sample Preparation Procedure

for Spiked Samples

CopyrightWatersCorporation2001Cuppett

Monoisotopic Masses

Benzodiazepines

Clonazepam

315.0411 m/z Diazepam

284.0716 m/z Flunitrazepam

313.0863 m/z Lorazepam 320.0119 m/z

Oxazepam

286.0509 m/z Temazepam

300.0666 m/z Triazolam

342.0439 m/z

N

N N N

Cl Cl

C H3 N

N CH3 O

Cl

N

N+ N O-

O CH3 O

F

N N

H O

OH Cl Cl

H

N N

H O

OH

Cl N

N O

OH CH3

Cl N+ N

NH O-

O

O

Cl

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Export Data from MassLynx into ACD

Export spectrum directly from MassLynx spectrum review window

into ACD/ SpecManager MS module

The ACD spectrum window provides further opportunities to

process data

Once the spectrum is imported into ACD/SpecManager, a structure is

integrated with the spectra using the integrated drawing package.

Thresholding can used to remove low intensity background noise.

From the spectrum window, the data is entered into a searchable

database along with the appropriate user data.

Add Spectrum to Database

Creating a UV and MS Database

4Individual benzodiazepine standards were analyzed under the conditions given previously

– All MS data (3 cone voltages used to enhance descriptive fragmentation for each compound) were entered into one comprehensive database using the procedure presented below

– Following a similar procedure, UV data were also incorporated into the database

4Chemical structures were incorporated into both the UV and MS library entries using the integrated drawing package

Manually Searching Data in the Database

4Ways to search data By spectra By structure By substructure By individual fragment

peaks By user-defined text By single DB or multiple

DB

Database Searching Capabilities

Once the database is populated, there are several ways in which the data may be accessed and searched.

Creating UV and MS Libraries

CopyrightWatersCorporation2001Cuppett

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Manual Interpretation of Low Concentration Samples

Little Information Available from TICs at Low Concentrations

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time

-1 100

% 19 100

% 14 100

% 20 100

%

Total ion current chromatograms (TIC) produced in the analysis of spiked serum (25 pg of each benzodiazepine on column). Little information is gained from TICs. By visualizing data with the Map function in MassLynx, it is possible to pick peaks out of the noise.

30 VTIC

50 VTIC

70 VTIC

PDATIC

25 pg on column

Pulling Peaks Out of the Noise

Using the Map function in MassLynx, it is possible to manually scroll through individual m/z ratios collected during a scan run for each function. Through this functionality, peaks may be picked out that are not evident in the TICs. For example, with a cone voltage at 70 V, a peak at 193 m/z was found around 16 minutes.

Extraction of “Hidden Peaks”

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time

3 100

%

100 150 200 250 300 350 400 450 500 550 600m/z 0

100

% 193

109154

125 132

156 175

222

194 228 230285

295325 351 417420459 520 516522554592

597

Extracted Ion Chromatogram 193 m/z Combined Mass Spectrum

Once individual masses are distinguished from the noise, extracted ion chromatograms can be created and used to generate mass spectra. The mass spectra can then be searched against MS libraries for identification. However, if the noise is high, extraneous peaks may diminish the utility of automated searches.

Ion Searches

In cases where manual interrogation of data is necessary, software that facilitates the process can translate into great time savings. ACD software provides the ability to easily ascertain the relative intensities of all the peaks and perform searches against MS libraries of selected peaks based on their relative intensities.

Relative Intensities Ion Search

Parameters

Ion Search Results

Match Found in Database Unknown Spectrum Ion Search Result in Library Comparison of Unknown Spectrum with Match Found in Library The manual selection of peaks and use of relative intensities for searches allows otherwise “unmatchable” spectra to be found in the database. Manual comparison of the library spectrum from the database and the unknown spectrum is then used to assist in the determination of the identity of the compound.

CopyrightWatersCorporation2001Cuppett

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Export Data from MassLynx into ACD

220 240 260 280 300 320 340 360 380 400nm

0 100

%

Benzo_SPE_Serum_25ng_051701_2 596 (10.040) Cm (593:598) 4: Diode Array 3.77e4 218

252

310

220 240 260 280 300 320 340 360 380 400m/z

0 100

%

Benzo_SPE_Serum_25ng_051701_2 667 (10.005) Cm (665:673-(636:648+686:693)) 3: Scan ES+

1.82e7 268

239

211 212 221 227

238 240

241 267

249 257 269

314 270286 300

287301315

325 335346347359365377381394

PDA and MS Data from MassLynx

Data is exported out of MassLynx into ACD/SpecManager. Thresholding is used to remove extraneous background

peaks from the MS spectrum.

The use of multi-spectral data can provide multiple reference points from which to verify compound identity. This can lead to increased confidence in results. Here we show how multi-spectral data can be used with the structure-based database.

LC/UV/MS run: 25 ng of each benzodiazepine standard on column

Searching the Database with Multi-Spectral Data

Search Using MS Data

Number of library matches PDA matches reflected in List A

MS matches reflected in List B Similarity between unknown spectrum and library spectrum Search Using PDA Data

Intersect List A and List B to confirm matching hits from the PDA and MS searches

1 overlapping hit from each library:

Flunitrazepam at 70 V

Multi-Spectral Data Using

Autofragmentation

The identified MS spectrum may be further evaluated using ACD/SpecManager’s Autofragmentation algorithm. Using a proposed structure, the software fragments the molecule and compares the fragments to the spectrum.

Proposed fragment

Percentage of spectrum assigned fragments

CopyrightWatersCorporation2001Cuppett

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Summary

4 Structure-based LC/MS database can provide useful tools for the analysis of unknown samples

– Peak searching for samples containing high background levels against commercial or in-house databases

– Library searching of both UV and MS spectra; higher degree of confidence when orthogonal results coincide

– Autofragmentation process assists in the analysis of fragment ions in MS spectra

– Chemical structure and substructure searching using prior knowledge can provide a means by which to analyze spectra

CopyrightWatersCorporation2001Cuppett

References

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