Presentation Outline

LC/MS/MS
Introduction and
Applications
Crystal Holt
LC/MS Product Specialist
Varian Inc.

Polarity, MW and Volatility

Introduction to LC/MS

Polarity, MW, Volatility

Instrumentation Components
MS/MS

Applications

Gabapentin
Benzodiazapines
Amphetamines
Carboxy-THC

LC/MS Triple Quad

Components

Overlap: GC/MS
or LC/MS

Solvent Delivery
System
O.D. Sparkman and F.E. Klink, "Chromatography/Mass Spectrometry: Principles
and Practice", American Chemical Society Continuing Education Courses, 2001.

AutoSampler

MS

LC/MS/MS Analyzer
Q1
Mass Analyzer

MS/MS whats that?

MS/MS is when you have more than one
step of mass filtration/separation and an
additional step of fragmentation.
MS/MS is used to give an additional level
of confirmation to an analysis or when very
specific results are required.
MS/MS can also help determine the
molecular structure of a molecule.

Q2
Collision Ce
Ionization
Source
Detector
Vacuum System

Q3
Mass Analyzer

MS/MS in a Triple Quad

Step 1 Ionization
GCMS

or LCMS

Triple Quad

Step 2 Isolation
Select

mass to
analyze using Q1.
Removes all other ions
that we arent
interested in.

Triple Quad

Triple Quad

Step 3 Fragmentation

Excite

ions in Q2 and
smash them into Ar
Collision Induced
Dissociation (CID)
The original ion is
smashed into smaller
pieces.

Use

the third
quadrupole to select
fragment ions.
Pass those product
ions to the detector for
quantitation.

Triple Quad SRM

Q2- Curved
Collision Cell
MRM or SRM
First transition

Q1

Step 4 Detection

Q3
Q1

Q2- Curved
Collision Cell

Q3

Example with a second transition (different precursor and fragment ions)

Q1

Q2- Curved
Collision Cell

Q3

MRM - often used to describe Multiple Reaction Monitoring.

SRM preferred term. Selected Reaction Monitoring (not single).
Relating to data acquired from specific product ions corresponding
to m/z selected precursor ions.
(IUPAC recommendations, www.msterms.com).

Why MS/MS
for LC/MS?

Limitation of API Interfaces

EI Full Scan MS of Reserpine

Atmospheric Pressure Ionization (API) dominate

LC/MS
(ES, ESI, APESI)
Chemical Ionization (APCI)
Photoionization (APPI)

100

All API mechanisms are soft

50

Electrospray

EI-GC/MS produces a wide range of ions

Information Rich
195

O
N

HN

212

O
O

Minimal

fragment ions formed during ionization

Typically only molecular ion information
Pseudo- or quasi-molecular ion(s)
Minimal qualitative information for confirmation
Adduct ions (Na, K, etc) are not useful for
confirmation

FullMS
Scan
MS of Reserpine
ESI (+)ESI
Full(+)
Scan
of Reserpine

141
77

265

301

608
413

448

0
40
90
(mainlib) Reserpine

140

190

240

290

340

390

440

490

540

Electron Ionization spectrum from NIST; probe sample introduction

ESI (+) MS/MS of Reserpine

(M+H)+

After MS/MS dissociation

More ions = more information

API-LC/MS creates very few ions

Limited information

O
O

359 381

109
226

395

251

Na
adduct

Product Ion Scan Spectrum

590

Advantages of MS/MS

Consumables

MS/MS offers increased selectivity for dirty

samples. It helps the users look for small
amounts of sample that would otherwise be
hidden by all the background noise.

Same Molecule, MS Vs. MS/MS

MS/MS capabilities cost more due to additional hardware and software

requirements in a transmission quadrupole, but often times the
performance is worth the price increase.

Applications

Analysis of
Gabapentin by
LC/MS/MS

What is Gabapentin?

Gabapentin Structure

Gabapentin, an analog of the neurotransmitter

GABA, is used as an anticonvulsant in the treatment
of epilepsy.
It was approved by the FDA as an anti-seizure
medication but has many controversial off-label uses,
such as in the treatment of bipolar disorder, social
anxiety disorder, obsessive-compulsive disorder and
insomnia.
Gabapentin is also widely used as a pain reliever and
was one of the 50 most prescribed drugs in the
United States in 2003 due to its mild side-effect
profile.
Gabapentin may increase the effect of other drugs
such as antidepressants, alcohol and pain relievers.

Chemical structure of Gabapentin,

(1-(aminomethyl)-cyclohexaneacetic acid)

Gabapentin Extraction Methodology

Methodology:

Procedure:

The extraction of Gabapentin

from biological specimens is
performed using the principles of
protein precipitation by acetonitrile
(without a clean up step) and
detection by LC/MS/MS.

Accurately measure 1.0 ml of

standard, quality control and case
samples.
Add 10 ul of internal standard (final
concentration = 10 ug/ml) to each
tube and vortex.
Aliquot 50 ul of each standard,
quality control and case sample
into new tubes.
Add 400 ul of acetonitrile to each
tube and vortex.
Centrifuge for 10 minutes.
Decant supernatant to a clean
tube.
Evaporate the supernatant to
dryness.
Reconstitute the samples with 200
ul of Methanol: Water (60:40).
Vortex.
Transfer the liquid to labeled
autosampler vials with inserts.
The samples are now ready!

Drugs to be determined
by this method:
Gabapentin, Baclofen (IS),
Carbinoxamine (Alternate IS)

Instrumentation:

Liquid Chromatograph with

Tandem Mass Spectrometer-Mass
Spectrometer Detector
Column: Varian Pursuit Diphenyl
(DP) Column Dimensions: 3um,
50mm x 2.0mm

Gabapentin MS Parameters

Conditions
MS Conditions:

HPLC Conditions:

Ionisation Mode: ESI positive

Scan Time: 1.0 s
Drying Gas: 350 C at 25 psi
Collision Cell Pressure: 2.0 mTorr

Column: Pursuit Diphenyl 50mm

x 2.0mm 3u
Guard Column: Pursuit Diphenyl
2.0mm I.D., 3u
Solvent A: Water
Solvent B: Methanol
Flow Rate: 200L/min
Injection Volume: 25L

Compounds

Capillary
Product Collision
Voltage Precursor Ions
Energy
(V)
(V)
Ion (m/z) (m/z)

Gabapentin

65 172

154

-26.50

Gabapentin
Baclofen
(IS)

65 172

137

-29.00

65 214

151

-36.50

LC Program:
Time (min) %A
0:00
60
1:00
60
1:30
5
3:30
5
3:35
60
5:00
60

%B
40
40
95
95
40
40

Gabapentin Calibration Curve

(1.0 ug/ml 25 ug/ml)

1.0 ug/ml extracted Gabapentin (left);

Baclofen - IS (right)

G A B A P E N T I N
C u r v e
y
=

MCoun ts BACLOFEN (IS) 1.0 ug ml

std.xms 151.0
(214.0>151.0)

kCounts 1.0 ug ml std.xms

172.0>137.0
1.0 ug ml std.xms
500
172.0>154.0

F i t :
L i n e a r ,
+ 1 . 0 7 0 8 0 6 x

R e p l i c a t e s1

O r i g i n :

F o r c e ,

W e i g h t :

N o n e

R e s p .
F a c t .
R S D :
C o e f f .
D e t . ( r 2 ) :

6 . 9
0 . 9

2 . 5

1.00

400

2 . 0

0.75

300
1 . 5

0.50

100

0.25

0.00

Peak Size / PS Std.

200

1 . 0

0 . 5

0.5

1.0

1.5

2.0

2.5
minutes

0.5

1.0

1.5

2 .0

2.5
minutes

0 . 0
0 . 5
A m o u n t

1 . 0
A m t .

S t d .

1 . 5
( U G / M L )

2 . 0

RSD = 6.9%; r2 = 0.999

Gabapentin Conclusion

LC/MS/MS is utilized in the analysis of gabapentin

because it allows for a simple, cost-effective clean
up, requires no derivatization of the drug and
provides accurate results with excellent sensitivity.
This application demonstrates that the Varian 1200L
triple quadrupole LC/MS/MS can identify and quantify
gabapentin in postmortem specimens with the use of
two MRM transitions.
Varian Inc. would like to provide a special thank you
to Sara Kegler and Dan Anderson from Los Angeles
County Department of Coroner for the validation of
both the extraction procedure and the instrument
parameters.

Analysis of
Benzodiazepines
by LC/MS/MS

2 . 5

Introduction to Benzodiazepines

Benzodiazepine Experimental Conditions

Benzodiazepines are central nervous system

depressants.
The first benzodiazepine, chlordiazepoxide
(Librium) was discovered in 1954 by the
Austrian scientist.
In 1963 approval for use was given to diazepam
(Valium) - a simplified version of Librium primarily to counteract anxiety symptoms.
Currently, fifteen members of this group are
marketed in the United States, and about 20
additional benzodiazepines are marketed in
other countries.

Benzodiazepine
Mass Spec
Details

Capillary

Q1

Q3

CID

Collision

Analyte

Energy

73.0Volts 271.0

140.0

-24.0Volts

73.0Volts 271.0

165.0

-24.0Volts

Nordiazepam

70.0Volts 285.0

193.0

-24.0Volts

70.0Volts 285.0

154.0

-19.5Volts

70.0Volts 286.0

222.0

-18.5Volts

70.0Volts 286.0

121.0

-23.0Volts

61.0Volts 287.0

241.0

-10.5Volts

61.0Volts 287.0

269.0

-12.0Volts

69.0Volts 290.0

226.0

-18.5Volts D-4 7-Aminoclonazepam

69.0Volts 290.0

121.0

-23.0Volts

69.0Volts 290.1

198.0

-24.0Volts

69.0Volts 290.1

154.0

-19.5Volts

72.0Volts 292.0

246.0

-10.5Volts

72.0Volts 292.0

274.0

-12.0Volts

60.0Volts 301.0

255.0

-18.5Volts

60.0Volts 301.0

283.0

80.0Volts 309.0

281.0

-23.5Volts

80.0Volts 309.0

205.0

-36.5Volts

85.0Volts 314.1

286.0

-18.5Volts

Diazepam

7-Aminoclonazepam

Oxazepam

D-5 Diazepam

85.0Volts 314.1

268.0

84.0Volts 316.0

270.0

-20.5Volts

84.0Volts 316.0

214.0

-36.0Volts

57.0Volts 321.0

275.0

-18.0Volts

57.0Volts 321.0

303.0

-16.0Volts

Column: Varian C18 Taxsil

100X2mm, 3u
Flow Rate: 0.3 mL/min
A: 5mM Ammonium Formate
B: CAN
Time (min) %A
%B
0:00
70
30
0:30
70
30
10:00
50
50
10:30
70
30
15:00
70
30
Injection Volume, 10 uL

Chromatogram
of Benzo Mix,
250 ng/mL
Calibrator

ESI Positive Mode

Argon pressure: 2.0 mtorr
Q1/Q3 peak width: 1.0/1.0
amu

Samples

Protein Precipitation
Extraction Method
Complex Matrix
Reconstituted in 50:50
MeOH:Water

MCounts271.0>140.0 [-24.0V]
10

Nordiazepam

0
MCounts285.0>193.0 [-24.0V]
50

Diazepam

0
MCounts286.0>222.0 [-18.5V]
50
0
MCounts287.0>241.0 [-10.5V]
0.0
MCounts290.0>226.0 [-18.5V]
2
0
MCounts290.1>198.0 [-24.0V]
20
0
MCounts292.0>246.0 [-10.5V]
4
0
MCounts301.0>255.0 [-18.5V]

D-5 Oxazepam

Temazepam

20
0
MCounts309.0>281.0 [-23.5V]
40
0
MCounts314.1>286.0 [-18.5V]

-10.5Volts

Triple Quadrupole LC/MS/MS

Mass Spec
ESI Positive
conditions:
LC Conditions:

Alprazolam

D-5 Alprazolam

-22.0Volts
Clonazepam

0
MCounts316.0>270.0 [-20.5V]
4
0
MCounts321.0>275.0 [-18.0V]
4

7-Aminoclonazepam

Oxazepam

D-4 7-Aminoclonazepam

D-5 Diazepam

D-5 Oxazepam

Temazepam

Alprazolam

D-5 Alprazolam

Clonazepam

Lorazepam

Lorazepam

2.5
5.0
7.5
10.0
12.5
minutes
Seg 1, Time: 0.05-15.10, Scan Functions: 28
2031
4107
6182
8257
10333 Scans

Benzodiazepine Summary

Representative Calibration Curves (10-1000ng/mL)

7-Aminoclonazepam
Curve Fit: Linear,
y =
+1.058993x

Replicates
32 2

Origin: Include,
-0.006779

Weight: None

Resp. Fact. RSD:

Coeff. Det.(r2):

8.179%
0.9993

Peak Size / PS Std.

R(2) = 0.999

RSD = 8.17

0
1

2
3
Amount / Amt. Std. (ng/mL)

Clonazepam
Curve Fit: Linear,
y =
+1.054153x

Replicates
32 2

Origin: Include,
+0.013111

Weight: None

Resp. Fact. RSD:

Coeff. Det.(r2):

5.639%
0.9978

Peak Size / PS Std.

R(2) = 0.997

RSD = 5.64

Excellent sensitivity
Quantitation limits will be below 10ng/mL for all
compounds
Excellent quantitation can be achieved using a select
few internal standards
All 8 drugs can be run simultaneously and many
more can be incorporated as to your needs
The LC program has a 15 minute run time. This can
be reduced greatly if separation is not required.
Additional sensitivity can be achieved through use of
time segments

0
1

2
3
Amount / Amt. Std. (ng/mL)

Amphetamines Experimental Conditions

Analysis of
Amphetamines
by LC/MS/MS

Methamphetamine: MS/MS Breakdown

Methamphetamine Calibration Curve

C al ibra tion C u rve R e por t

C al ib ra tion C u rve R e p or t

File : e :\ n o r ch e m \ a m p s \a m p h e t a m in e sd a t a m u lt ip lie r2 . m t h
D e t e c to r : 1 2 0 0 M a s s S p e c , A d d re s s: 4 2

File : e :\ n o r ch e m \ a m p s \a m p h e t a m in e sd a t a m u lt ip lie r2 . m t h
D e t e c to r : 1 2 0 0 M a s s S p e c , A d d re s s: 4 2

M eth am phe tam ine

C u rv e Fit : L in e a r,
y =
+ 0.031 914x
R ep lic ate s

93

O rig in : I g n o r e ,
+0 . 2 5 7 2 7 1

W e ig h t : N o n e

M e tham ph eta m in e d 5
R e sp . F a ct . R S D : 5 . 3 1 8 %
C o e f f. D e t .( r2 ) : 0 . 9 9 7 4 1 9

C u rv e Fit : L in e a r,
y =
+ 1.000 000x

O rig in : I n c lu d e (I g n o r e ),
+0.0

W e ig h t : N o n e

R e sp . F a ct . R S D : 0 . 0 0 0 0 %
C o e f f. D e t .( r2 ) : 1 . 0 0 0 0 0 0

R ep lic ate s

24
M
15 0

30 0

12 5

25 0

10 0

20 0

15 0

Peak Size

Peak Size / PS Std.

75

10 0

50
50

0
25
2 50 0

5 0 00

75 00

A m ou nt / A m t. S td .

Methamphetamine 5-10,000 ng/mL

Chromatographic data of Amphetamines

0 .99 9 92 5

0.9 9 99 50

0. 9 99 97 5

1 . 00 00 0 0

1. 0 00 0 25

1 .00 0 05 0

1.0 0 00 75

Am o un t

Methamphetamine d5 IS

Amphetamine Summary

Spiked: 50 ng/mL in urine

0. 9 99 9 00

Excellent sensitivity at the level of 5ng/mL

Good separation from a column packed with a
fluorinated phenyl phase
Detection limits will be at least the low ppb levels
for all compounds
All 6 drugs can be run simultaneously in a 6 min.
run
Good linearity of calibration curves ranging from
5-10,000 ng/mL

Carboxy-THC Experimental Conditions

Analysis of
Carboxy-THC
by LC/MS/MS

Chromatograms Carboxy-THC

ESI Negative.
LC Conditions:

Column: Varian Pursuit Diphenyl

50X2mm, 3
Flow Rate: 0.2 mL/min
A: Water
B: Methanol
Time %B
0:00
40
0:30
40
1:00
95
3:00
95
3:01
40
6:30
40
Injection Volume, 20 L

Mass Spec
conditions:
Argon pressure: 2.0 mtorr
Q1/Q3 peak width: 1.0/1.0
amu
Transitions:

343.50 -> 299 CE 17.5

352.50 -> 308 CE 20.5

Samples

In a complicated matrix
solution
Standard Solutions prepared
by serial dilutions in DI water
50 L IS added to 1 mL of
sample

Carboxy-THC Calibration Curve Report

Carboxy-THC 10 ng/mL

Carboxy-THC 1 ng/mL

Carboxy-THC 1-1000
ng/mL

Carboxy-THC d9 - IS

Carboxy-THC Sample Report Example

Carboxy-THC Summary

Excellent sensitivity at the 1 ng/mL

Detection limits in the sub ppb levels
Linear calibration curve from 1-1000 ng/mL
R2 value of 0.998
Run time of only 6.5 minutes
Sample prep does not require derivatization
simply dilute urine in water and inject!