Mass Spectrometry

Solomon Tesfalidet, Analytical Chemistry, 2016

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 Mass Spectrometry
A Technique for studying the masses
of atoms or molecules or fragments
of molecules.

Gaseous phases are:

• Ionzed
• Accelerated by an electric
field
• Separated according to
their mass-to-charge ratio
m/z
• If all charges are +1, the
m/z is numerically equal to
the mass

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Principle

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4
 Magnetic Sector MS
Electron Ionization
Electrons emitted from a hot
filament are accelerated through a
potential of 70 V before they
interact with incoming molecules

Analyte molecules (M) absorb

as much as 12-15 electron volts,
which is enough for ionization

The magnetic field allows ions of

selected m/z to pass from the ion
source to the detector

The tube is maintained under

high vacuum (ca 10-5 Pa)
5
1 atmosphere = 101325.01 pascal
Electron ionization (EI) Chemical Ionization (CI)
CH4+ + e-  CH4+. + 2e-
M + e-  M+ + e- + e- CH4+. + CH4  CH5+ +.CH3
70eV 55eV 0.1 eV CH4+.  CH3+ + H
CH3+ + CH4  C2H5 + H2
Molecular ion
CH5+ + M  CH4 + MH+

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 Electrospray Ionization

• Little fragmentation in electrospray

• Fragmentation is increased by Collisionally Activated

Dissociation CAD (by adjustment of skimmer potential)
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Quadrupole Mass Spectrometer • Ions are accelerated through a
potential of 5-15V before
entering the quadrupole filter

• Only ions of a particular mass-

to-charge ratio reach the
detector

• Ions of different masses are

allowed to reach the detector by
rapidly varying the applied
voltages

• A detector of choice for

chromatography

• Cheap

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 Tandem Mass Spectrometry : Triple Quadrupole

Increased selectivity and

s/n ratio

Selected Reaction Monitoring (SRM)

• The molecular ion (precursor ion) is selected by the first quadrupole (Q1)
• The selected ion is fragmented using the second quadrupole (collision
cell) producing product ions (Q2)
• Selected product ions analyzed using the third quadrupole (Q3)

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 Ion-Trap Mass Spectrometer
• Separates ions by capturing
A compact device that is well them within a circular
suited as a chromatography electrode, where they orbit
detector until they are ejected by
variation in voltage
• Can filter out background
while the ion of interest is
retained in the trap before
being fragmented and
ejected
• Scans from m/z 10 to 650
can be conducted eight
times per second

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Time of Flight Mass Spectrometer TOF

• High acquisition rate (102

to 104 spectra/s)

• Capable of measuring very

high masses (m/z = 106)

• Requires lower operating

pressure than quadrupole
(10-12 versus 10-9 bar)

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Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
For analysis of trace elements
ICP-torch

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Calibration methods for determination of concentrations:

• External calibration curve: (often not so useful)

• Matrix matched calibration (when possible)

• Standard addition (commonly used)

• Internal Standard (added to a sample in known concentration to facilitate the

qualitative identification and/or quantitative determination of the sample)

• Isotope Dilution (if isotopically labeled standards are available)

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 Why Isotope Dilution? How does it work?

Fly dilution (Jackson Entomologist 1933)

+ =

Marked flies in sample = Total marked flies in circle

Umnarked flies in sample Total unmarked flies in circle

10 = 3
Unmarked flies in sample 6
6 X 10 = 20
Unmarked flies in sample =
3

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 Hair, 1-3 cm close to the scalp SPE
Determination of Rinse twice, dichloromethane and cut into 1-2 mm sections
Anabolic Steroids 50 mg hair
in hair: GC-MS/MS + 1mL NaOH (1M) + D3-testosterone (20pg/g hair)
Incubate for 15 min, 95 °C
+1ml HCl (1M) + 2mL phosphate (0.1M) = pH 7
Extraction on C18 - column
Elute with 5 ml MeOH
Evaporate, N2(g), 50 °C
+ 1 mL phosphate buffer (0.1M, pH 7)
Adjust pH (9-10) with 7% potassium carbonate
Extract with 5mL diethyl ether, shake 10min
Centrifugate 10min, 3000 rpm
Evaporate, N2(g)
+ 50 µL MSTFA-NH4I-DTE (1000:2:4 v/w/w)
GC-MS/MS
EI
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 Determination of Nandrolone and fluoxymesterolone in hair

Nandrolone Fluoxymesterolone

Gambelunge et.al., Biomed. Chromatogr. 2007, 21, 369-375

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Determination of
Cocaine in Hair: MeOH-extraction
GC-MS/MS Hair, 1-3 cm close to the scalp
Rinse twice, dichloromethane, dry and cut into 1-2 mm sections
50 mg hair
+ 1mL MeOH + D3-testosterone (10pg/g hair) IS
Sonicate for 2 hours (56 °C), thermostatic bath (56 °C) overnight

+1ml HCl (1M) + 2mL phosphate (0.1M) = pH 7

Centrifugation 15 min, 3000 rpm
Decant the MeOH phase, evaporate to dryness with N2(g)
Derivatize: + 50 µL MSTFA-NH4I-DTE (1000:2:4 v/w/w)
cocaine evaporate, N2(g)
+ 1mL MeOH

EI GC-MS/MS

Gambelunge et.al., Biomed. Chromatogr. 2007, 21, 369-375

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 Cocaine and Benzoylecgnonine in hair

Cocaine
Benzoylecgnonine

Gambelunge et.al., Biomed. Chromatogr. 2007, 21, 369-375

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Determination of organic and inorganic Hg in blood
Mercury in thimerosal (preservative in
manufacturing vaccines)
Timerosal
What happens with it
M = 404.81 g/mol
in our body?
MHg = 200.59 g/mol

C9H9HgNaO2S
Thimerosal decomposes to
Eth-Hg and thiosalyicylate

How do we determine Eth-Hg in our body?

Jairo et.al., Talanta 80 (2010) 1158,1163
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Determination of organic and inorganic Hg in blood (cont..)
Total Hg Blood Hg-speciation

Determination of inorganic
and organic Hg in blood 50 times dilution
50 times dilution
using HPLC-ICP-MS with 0.5% HNO3
with 0.5% HNO3
10 mL in a test tube 250µL in a test tube
+ 25µgL-1Rh (IS) + 4.75mL 0.1%(v/v) HCl
+ 0.05%(m/v) L-cysteine
+ 2-mercaptoethanol
+ 1mgL-1 Au
Sonication, 15min
50 times dilution
+ 0.005% Triton X-100
with 0.5% HNO3

ICP-MS Centrifugation, filtration

HPLC-ICP-MS

Jairo et.al., Talanta 80 (2010) 1158,1163

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Determination of organic and inorganic Hg in blood (cont..)

Chromatograms showing the

separation of mercury species
in: (a) standards containing
10 μg L−1 of each mercury
compound and (b) human
blood (volunteer from fish-
eating communities of the
Amazon region).

Jairo et.al., Talanta 80 (2010) 1158,1163

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Determination of inorganic and organic Hg in blood from a rat exposed to Eth-Hg

Chromatograms showing the separation of

mercury species in blood of:

(a)rat before ethyl mercury exposure (control)

and

(b)exposed to 100 μg kg−1 day−1 of ethyl

mercury during 3 days.

Et-Hg is converted invivo to ino-Hg

“Extremely relevant finding in evaluation of thimerosal exposed populations”

Talanta 80 (2010) 1158, 1163
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