gc/ms fundamentals: what are gc/sq, tq and …...gc/ms fundamentals: what are gc/sq, tq, and q-tof...

GC/MS Fundamentals:What are GC/SQ, TQ, and Q-TOF and When to Use Them?

Angela Smith Henry, Ph.D.Applications Chemist, GC and MS Supplies, Wilmington, DE

Bruce Quimby, Ph.D.Applications Chemist, MSD, Wilmington, DE

Kai Chen, Ph.D.Applications Chemist, MSD, Santa Clara, CA

Jessica WestlandApplications Chemist, Sample Prep, Wilmington, DE

July 25, 2019

Agilent Technologies For Research Use Only1

2 Agilent Technologies For Research Use Only

Objectives

Overview of how Agilent’s GC/MS systems work:• GC/single quad (GC/MSD SQ)

• GC/triple quad (GC/MS TQ)

• GC/quadrupole time-of-flight (GC/MS Q-TOF)

Overview of how customers use each type to solve their analytical problems• Example: Pesticides in essential oils

SQ GC/MSD

Separate

compounds

Create ions and

fragmentsSeparate ions Detect ions

Vacuum

GC Ion source Quadrupole High-energy dynode,

and electron multiplier

Column

SQ GC/MSD

Separate

compounds

Create ions &

FragmentsSeparate ions Detect ions

Vacuum

GC Ion source Quadrupole High-energy dynode,

and electron multiplier

Column

40 80 120 160 200 240 280 3200

318During the run, quadrupole is (for example)

scanned from 35 m/z to 500 m/z at 3.2

scans/sec. Signal that reaches the detector is

recorded in the data file.

Create ions and

fragments

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

50 6275 87 99

105123

SQ GC/MSD: Example Spectrum at 24.065 Minutes

rmalized

Scan data is processed to create:

• Total Ion Chromatogram (TIC)

• Base Peak Chromatogram (BPC)

• Extracted Ion Chromatograms (EIC)

• Spectra for library searching

• Spectra for unknown identification

40 80 120 160 200 240 280 3200

Spectrum

5 10 15 20 25 30 35 40

23.8 23.9 24.0 24.1 24.2 24.3

Ion 318

Ion 246

Ion 316

Ion 248

m/zTime (min)

Time (min)

p,p’-DDE

SQ GC/MSD: Processing SQ Scan Data

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

50 6275 87 99

105123

267281

(main lib) p,p'-DDE

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

75 8799

NIST Library

Spectrum

p,p’-DDE

Library

Match Score

Unknown

Spectrum

SQ GC/MSD: Library Searching SQ Scan DataSearching unknown spectra

against NIST or other libraries

is widely used for identification

of compounds.

Spectral deconvolution software

from NIST (AMDIS) and now

MassHunter SureTarget can be

used to produce spectra with

the ions from overlapping

matrix peaks removed.

40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

50 6275 87 99

105123

267281

318Full scan

spectrum

35–500 amu

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 3200

263 277

monitoring 8

Ions instead of

SIM: Single Ion MonitoringDuring chromatographic

run, quad is programmed

to monitor a small number

of ions for specific

analytes in specific time

ranges.

More time is spent

measuring each ion vs

This gives ~10 x better

signal-to-noise and better

area precision. However,

ability to search spectra

for unexpected unknowns

is lost.

20 22 24 26 28 30

TIC Spinach

Extract

EIC 246 amup,p’-DDE

The p,p’-DDE peak is visible in

this spinach extract, but the

baseline shows that the SQ

has only limited selectivity over

other matrix compounds that

also have m/z 246 as a

fragment.

This limits the ability of the SQ

to find traces of pesticides in

extracts with high levels of

matrix interferences.

matrix interferences

at m/z 246

SQ GC/MSD: Limited Selectivity of SQ

SQ GC/MSD: Summary

Most widely used GC/MS technique• Least complicated

• Relatively rugged

• Alternative to traditional GC detectors in many applications

Modes of use• Quantitative analysis (how much is in there?) using target ion and 1–3 qualifiers

• SIM: best S/N (detection limit), but limited number of analytes and no spectra for identifications

• Scan: ~10 x poorer S/N, unlimited number of analytes and spectra for identifications

• Qualitative analysis (what is in there?)• Scan: Searching of deconvoluted spectra against libraries like NIST, Agilent RTL libraries, or others

• Retention time versus response chromatogram like any GC detector

Limitations• Selectivity over interferences from matrix

• Selectivity over some GC detectors (uECD, FPD, NPD, SCD, etc.)

• Unit mass resolution limits spectral interpretation of unknowns

extractor source

HES source

Agilent GC TQ Systems

MS/MS requires multiple mass filters in series

TQ GC/MS: What is MS/MS Analysis?

Selection of

precursor ion

Fragmentation

of precursor ion

(Collision cell)

Selection of

fragment ions

Typical analysis = Detection

Carrier Gas He

Ion sourceMS1

mass filter

Hexapole

collision cell

mass filterDetector

Collision Gas N2

Quench Gas He

Step 1: Precursor ion of target passed by MS1. Contains fragment ions of mass 210 created from both analyte and interference molecules.

170 210 250 290

268 280165

MS1 lets only target ion

210 pass through

TQ GC/MS: Multiple Reaction Monitoring (MRM)

Ion sourceMS1

mass filter

Hexapole

collision cell

mass filterDetector

MS1: SIM

210 m/z

MS2: SIM

158, 191 m/z

Spectrum with background ions

Step 2: The collision cell generates ions from the mass 210 fragments from both analyte and interferences.

170 210 250 290

268 280165

150 170 190 210

210158 191

Collision cell breaks ion

210 into fragments

MS1 lets only target

ion 210 pass through

Spectrum with background

Ion sourceMS1

mass filter

Hexapole

collision cell

mass filterDetector

MS1: SIM

210 m/z

MS2: SIM

158, 191 m/z

Step 3: MS2 is set to filter for quantification and qualifier product ions only formed by the analyte (unique ions.)

MS2 monitors only characteristic fragments 158

and 191 created from ion 210, for quant and qual.

170 210 250 290

268 280165

150 170 190 210

210158 191

Collision cell breaks

ion 210 into fragments

MS1 lets only target

ion 210 pass through

Spectrum with background

Ion sourceMS1

mass filter

Hexapole

collision cell

mass filterDetector

MS1: SIM

210 m/z

MS2: SIM

158, 191 m/z

MRM (246→175)

Orange

Spinach

Ginseng

Cabbage

SIM (246)

S/N = 448

S/N = 456

S/N = 260

S/N = 446

S/N = 241

(All injections = 1 µL)

Comparison of SQ SIM and TQ MRM data: p,p’-DDE, 10 ppb, various matrices

Most widely used GC/MS technique for target analysis of pesticides• More complicated method development choosing precursor and product ions and collision voltages

• MRMs provide extremely high selectivity necessary in complex matrices

Modes of use• Quantitative analysis (how much is in there?)

• MS2 SIM similar to SQ SIM

• MRM using target ion and 1-2 qualifiers

• Qualitative analysis (what is in there?)• Scan: Searching of deconvoluted spectra against libraries like NIST, Agilent RTL libraries, or others

Limitations• Unit mass resolution limits spectral interpretation of true unknowns

• Limited number of MRMs per run

• Scan mode useful, but not quite as good as SQ in S/N or spectral match to NIST

TQ GC/MS: Summary

Q-TOF GC/MS

7200 Q-TOF

Extractor Source

7250 Q-TOF

HES Source

m ∞ 𝐭2

The kinetic energy, Ke, of ions as they are pulsed in the flight tube, where

m = mass v = velocity

The ion velocity equals the flight path (L) divided by (t) time

𝑲𝒆 =𝟏

𝟐𝒎𝒗𝟐

𝒗 = 𝑳/𝒕

𝒕= 𝟏/𝟐𝒎𝒗𝟐

𝒎 = 𝒕𝟐 𝟐𝑲𝒆 /𝑳𝟐 2𝐾𝑒 /𝐿2 is considered constant

Combine equations…

The higher the m/z for an ion, the longer it takes to transit

the flight tube and reach the detector

Q-TOF GC/MS: What is Time of Flight?

Detector

Reflectron

Flight tube

Ion source Detector

20 µsec – m/z 118

46 µsec - m/z 622

90 µsec - m/z 2421

1. Pulse ions every 100 microseconds.

2. Measure at detector each nanosecond.

3. 100,000 data points in each transient.

4. Sum 2000 – 10,000 transients into one spectrum.

Produces spectra with excellent ion statistics.

Transient—one packet of ions pulsed down

the flight tube and detected. Data from many

pulses, typically ~10,000 transients, are

summed to create a spectrum.

Q-TOF GC/MS: Transients

Detector

Reflectron

Flight tube

Ion source Detector

Q-TOF GC/MS: MS or TOF mode

Total ion transmission

through quadrupole

TOF – Separation of ions by time

No energy applied in collision cell

10117&8

Ionization

in source

Detection and

digitalization

TOF mode can provide the accurate mass of the molecular ion, when available.

Similar to EI scan mode spectra from a SQ.

(Base peak)

(Molecular ion)

170.2014

Q-TOF GC/MS: A typical spectrum in MS mode

The molecular ion in EI mode: loss of one electron, a radical cation, M+

Base peak: most abundant ion in spectrum.

Most fragment ions are even electron ions, except where rearrangements have occurred.

Single Quadrupole and TOF spectra will be slightly different due to the ion path length, TTI cut-off, etc.

<--[C6H13]+

<--[C5H11]+

<--[C4H9]+

Single Quad Scan Data

Q-TOF Data

C12H12

- 0.3 ppm Standard EI (70 eV)

NIST Search

Score: 87.2

1,3,7-trichloro-naphthalene

C10H5Cl3

- 106 ppm

Acquisition Time (min)

4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30 8.40 8.50 8.60

NIST Search

Score: 94.7

2,6-dimethylnaphthalene

• Good library match and accurate mass

confirm the identification

• Large mass error suggests different ID or

unknown

• Accurate mass MS/MS help elucidate the

candidate structure of true unknown

Q-TOF GC/MS: Untargeted Screening and Identification of Unknowns by Q-TOF

Broccoli

sample

Full spectrum acquisition

MS/MS CID@25 eV

Step 1: Confirm M+ Step 2: Confirm fragment ions

Step 3: Structure elucidation on

candidate

C8-H-Cl3-N2 turns out to be 2,4,5-Trichloroisophthalonitrile.

That is not NIST. Agilent Molecular Structure Correlator.

It is a degradation product of Chlorothalonil.

2.1 ppm

3.63 ppm

2.3 ppm

Low energy EI (15 eV)

Isotope pattern and mass accuracy are good

Q-TOF GC/MS: Confirming structures with low energy EI MS/MS andaccurate mass

Q-TOF GC/MS: Summary

Newest GC/MS technique available from Agilent• Provides, high-resolution, accurate-mass scan data with sensitive detection.

• High-resolution allows results in BOTH high selectivity over matrix AND sensitive detection

• High-resolution and accurate mass provides chemical formulas for spectral interpretation of true unknowns

• Easier method development than TQ

• Much higher selectivity than SQ but not as high as TQ

Modes of use• Quantitative analysis (how much is in there?)

• EICs using target ion and qualifiers

• Since all data is scan mode, unlimited number of analytes

• Qualitative analysis (what is in there?)• Screening for large number (>1000) of pesticides using accurate mass fragments

• Searching of spectra against libraries like NIST, Agilent RTL libraries, or others

• Spectral interpretation of true unknowns using accurate mass data

Limitations• High cost

• Bench space required

• Very large data file size, requires high powered data processing

Analysis of Pesticides and Environmental Pollutants in Essential Oils Using Multi-Platform GC/MSD, GC/TQ and GC/Q-TOF

Kai Chen

Agilent Technologies, Inc., Santa Clara

Bruce Quimby

Agilent Technologies, Inc., Wilmington, Delaware

Vivian Xianyu Chen

Agilent Technologies (Shanghai) Co., Ltd., Shanghai, China

GC/MSD: Use a retention time locked (RTL) spectral library of 950+ compounds and screen based on match scores of deconvoluted spectra.

GC/TQ: Initial screening of samples using scan mode (as above). Subsequent MRM analysis for trace level identity confirmation and quantitation.

GC/Q-TOF: Use an RTL library containing accurate mass spectra for 850+ compounds and screen using six principle accurate mass ions, fragment ratios and RT matching.

This multi-platform GC/MSD, GC/TQ and GC/Q-TOF approach not only provides comprehensive and flexible analysis methods, but also offers a more streamlined workflow using different platforms.

Using SQ, TQ, and Q-TOF to Examine Essential Oils for Pesticide Residue

Agilent Technologies For Research Use Only

Samples and Data Acquisition

Essential oils as model complex matrices

• Grapefruit oil

• Lemon oil

• Neroli oil

• Orange oil (Brazil origin)

• Orange oil (California origin)

• …

Mid-column backflush system

5977B GC/SQ: 1:10 dilution

7250 GC/Q-TOF: 1:50 dilution

7010 GC/TQ: 1:100 dilution

GC/Q-TOF

GC/MSD

Grapefruit Oil: Severe matrix interferences

+ TIC Scan Grapefruit.D

Counts vs. Acquisition Time (min)

3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5

Pyrimenthanil

Malathion

GC/SQ TIC

Grapefruit Oil

8.2 8.25 8.3 8.35 8.4

198.1026

199.1104

200.1127

183.0791

197.0947

Deconvoluted MSD

spectrum

Library

spectrum

Q-TOF TQMSD

Raw MSD

spectrum

Counts vs. Acquisition Time (min)

8.2 8.25 8.3 8.35 8.4

Component RT: 8.2928

Acquisition Time (min)

8.3 8.35C

nts 6x10

1.8198.1

Match score 96

Quantitation with TQ MRM

mode found Pyrimenthanil

was 29.4 ng/mL as injected

(100:1 dilution)

Pyrimenthanil Found In Screening By All Three Systems

SQ is overwhelmed by

interferences. No indication

of Malathion.

Q-TOF finds Malathion with good

RT match, fragment ratio score

and mass accuracy.

TQ scan screen also missed Malathion, but

MRM mode found it easily, with quant value =

1.26 ng/mL as injected (100:1 dilution).

Malathion: Very Large Interferences

Compound Name

GC/MSD GC/Q-TOF GC/TQ (MRM)

RT Diff

Frag Ratio

RT Diff

Mass Diff

RT Diff

(ng/mL)

Aniline 68 0.068 rejected after review n.d. n.d.

Pyrimethanil 96 0.023 98.05 0.015 3.68 0.012 29.41

Chlorpyrifos Methyl 79 0.000 99.92 0.005 1 0.006 3.33

Chlorpyrifos 93 -0.002 95.33 0.006 0.42 0.009 30.69

Imazalil 57 0.047 95.99 0.048 0.88 0.019 25.20

Pyriproxyfen 81 0.020 85.83 0.019 1.55 0.016 10.86

Malathion n.d. n.d. 82.15 0.014 0.17 0.011 1.26

Primiphos-methyl * n.d. n.d. n.d. n.d. n.d. 0.011 0.22

Oxadixyl * n.d. n.d. n.d. n.d. n.d. 0.021 0.36

Methidathion * n.d. n.d. n.d. n.d. n.d. 0.004 0.54

* quantified by the routine multi-residue MRM method on TQn.d. = not detected or identified

Identification and quantification results from grapefruit oil by multiple platforms

Results Summary Table

Summary

For discovery of pesticides present:

MSD or TQ Scan: Examine data file using:

• SureTarget spectral deconvolution and Agilent unit mass pesticide library.

Q-TOF Scan: Examine data file using:

• Accurate mass pesticides library (PCDL) with 850+ compounds via Find by Fragments in MassHunterQualitative Analysis

• SureTarget spectral deconvolution and Agilent unit mass pesticide library in Unknowns Analysis for compounds not in PCDL

For quantitation of pesticides present:

MSD Scan or SIM: Quant using ions with least interference for discovered pesticides in that matrix.

TQ MRM: Quant using MRMs with least interference for discovered pesticides.

Q-TOF: Quant using accurate mass EICs with least interference for discovered pesticides.

Thank you for your attention!

Any questions?

Pesticides in Essential Oils

Essential oils are concentrated liquids containing volatile aroma compounds from various

plants and are widely used in flavors and fragrances. Traces of pesticides are sometimes

present in these oils, resulting in the need to screen for them.

This analysis is challenging due to the large background interference presented by oil

matrices. Traditional GC/MS is limited in capacity to perform the task. This work

demonstrates the use of advanced techniques for pesticide screening in essential oils.

GC and MS Conditions Value

Columns (2 ea.)Agilent J&W HP-5ms UI, 15 m × 0.25 mm × 0.25

μm (P/N: 19091S-431 UI)

Inlet MMI, 4 mm UI liner with wool

Injection 1 or 2 µL cold splitless

Carrier gas Helium

Inlet flow (column 1) ~ 1 mL/min

PUU flow (column 2) column 1 flow + 0.2 mL/min

Oven program

60°C for 1 min

40°C/min to 170°C for 0 min

10°C/min to 310°C for 3 min

(Total run time: 20.75 min)

Backflush Conditions5 min (postrun). 310 °C (oven).

50 psi (aux EPC pressure). 2 psi (inlet pressure)

Transfer line temperature 280 °C

Ion source temperature 280 °C

Quadrupole temperature 150 °C

Chromatographic Parameters

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