LC/MS/MS Introduction and ApplicationsCrystal HoltLC/MS Product SpecialistVarian Inc.

Presentation OutlineIntroduction to LC/MS

Polarity, MW, VolatilityInstrumentation ComponentsMS/MS

ApplicationsGabapentinBenzodiazapinesAmphetaminesCarboxy-THC

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

Polarity, MW and Volatility

Overlap: GC/MS or LC/MS

LC/MS Triple Quad Components

Solvent DeliverySystem AutoSampler MS

LC/MS/MS Analyzer

Q2Collision Ce

Vacuum System

Ionization Source

DetectorQ3

Mass Analyzer

Q1 Mass Analyzer

MS/MS what’s 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.

MS/MS in a Triple Quad

Step 1 IonizationGCMS or LCMS

Triple Quad

Step 2 IsolationSelect mass to analyze using Q1.Removes all other ions that we aren’t interested in.

Triple QuadStep 3 Fragmentation

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

Triple QuadStep 4 Detection

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

Triple Quad SRM

MRM - often used to describe “Multiple” Reaction Monitoring.SRM – preferred term. Selected Reaction Monitoring (not single).Relating to data acquired from specific product ions correspondingto m/z selected precursor ions.(IUPAC recommendations, www.msterms.com).

Q3

Q2- Curved Collision Cell

Q1

MRM or SRM

Q1 Q2- Curved Collision Cell

Q3

Q1 Q2- Curved Collision Cell

Q3

First transition

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

Why MS/MSfor LC/MS?

Limitation of API InterfacesAtmospheric Pressure Ionization (API) dominate LC/MS

Electrospray (ES, ESI, APESI)Chemical Ionization (APCI)Photoionization (APPI)

All API mechanisms are “soft”Minimal fragment ions formed during ionization

Typically only molecular ion informationPseudo- or quasi-molecular ion(s)

Minimal qualitative information for confirmationAdduct ions (Na, K, etc) are not useful for confirmation

(mainlib) Reserpine40 90 140 190 240 290 340 390 440 490 540 590

0

50

100

77 109

141

195

212

226

251

265 301

359381395

413 448

608

O

O

OO

NHN

O

O

O

O

O

EI Full Scan MS of Reserpine

EI-GC/MS produces a wide range of ions“Information Rich”

Electron Ionization spectrum from NIST; probe sample introduction

ESI (+) Full Scan MS of Reserpine

API-LC/MS creates very few ionsLimited information

Naadduct

(M+H)+

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

After MS/MS dissociationMore ions = more information

Product Ion Scan Spectrum

ESI (+) MS/MS of Reserpine

Advantages of MS/MS

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.

Consumables

ApplicationsAnalysis of Gabapentin by LC/MS/MS

What is Gabapentin?

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.

Gabapentin Structure

Chemical structure of Gabapentin,(1-(aminomethyl)-cyclohexaneacetic acid)

Gabapentin Extraction MethodologyMethodology:

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

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

Instrumentation:Liquid Chromatograph with Tandem Mass Spectrometer-Mass Spectrometer DetectorColumn: Varian Pursuit Diphenyl(DP) Column Dimensions: 3um, 50mm x 2.0mm

Procedure: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!

Gabapentin MS ParametersConditionsMS Conditions: Ionisation Mode: ESI positiveScan Time: 1.0 sDrying Gas: 350 ˚C at 25 psiCollision Cell Pressure: 2.0 mTorr

HPLC Conditions:Column: Pursuit Diphenyl 50mm x 2.0mm 3u Guard Column: Pursuit Diphenyl2.0mm I.D., 3uSolvent A: WaterSolvent B: MethanolFlow Rate: 200µL/minInjection Volume: 25µL

LC Program:Time (min) %A %B0:00 60 401:00 60 40 1:30 5 953:30 5 953:35 60 405:00 60 40

Compounds

Capillary Voltage (V)

Precursor Ion (m/z)

Product Ions (m/z)

Collision Energy (V)

Gabapentin 65 172 154 -26.50

Gabapentin 65 172 137 -29.00 Baclofen (IS) 65 214 151 -36.50

1.0 ug/ml extracted Gabapentin (left); Baclofen - IS (right)

0.5 1.0 1.5 2.0 2.5minutes

0

100

200

300

400

500

kCounts 1.0 ug ml std.xms172.0>137.01.0 ug ml std.xms172.0>154.0

0.5 1.0 1.5 2.0 2.5minutes

0.00

0.25

0.50

0.75

1.00

MCounts BACLOFEN (IS) 1.0 ug mlstd.xms 151.0(214.0>151.0)

Gabapentin Calibration Curve (1.0 ug/ml – 25 ug/ml)

G A B A P E N T I NC u r v e F i t : L i n e a r , 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 : 6 . 9

C o e f f . D e t . ( r 2 ) : 0 . 9y = + 1 . 0 7 0 8 0 6 x

Peak Size / PS Std.

A m o u n t / A m t . S t d . ( U G / M L )0 . 5 1 . 0 1 . 5 2 . 0 2 . 5

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

2 . 5

R e p l i c a t e s1 1 1 1 1 1

RSD = 6.9%; r2 = 0.999

Gabapentin Conclusion

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

Introduction to BenzodiazepinesBenzodiazepines 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 Experimental Conditions

• Mass Spec conditions:

• ESI Positive Mode• Argon pressure: 2.0 mtorr• Q1/Q3 peak width: 1.0/1.0

amu

Triple Quadrupole LC/MS/MSESI PositiveLC Conditions:

• Column: Varian C18 Taxsil100X2mm, 3u

• Flow Rate: 0.3 mL/min• A: 5mM Ammonium Formate• B: CAN• Time (min) %A %B

0:00 70 300:30 70 30 10:00 50 5010:30 70 3015:00 70 30 Injection Volume, 10 uL

• Samples• Protein Precipitation

Extraction Method • Complex Matrix• Reconstituted in 50:50

MeOH:Water

Benzodiazepine Mass Spec Details

+ 57.0Volts 321.0 303.0 -16.0Volts

+ 57.0Volts 321.0 275.0 -18.0Volts Lorazepam

+ 84.0Volts 316.0 214.0 -36.0Volts

+ 84.0Volts 316.0 270.0 -20.5Volts Clonazepam

+ 85.0Volts 314.1 268.0 -22.0Volts

+ 85.0Volts 314.1 286.0 -18.5Volts D-5 Alprazolam

+ 80.0Volts 309.0 205.0 -36.5Volts

+ 80.0Volts 309.0 281.0 -23.5Volts Alprazolam

+ 60.0Volts 301.0 283.0 -10.5Volts

+ 60.0Volts 301.0 255.0 -18.5Volts Temazepam

+ 72.0Volts 292.0 274.0 -12.0Volts

+ 72.0Volts 292.0 246.0 -10.5Volts D-5 Oxazepam

+ 69.0Volts 290.1 154.0 -19.5Volts

+ 69.0Volts 290.1 198.0 -24.0Volts D-5 Diazepam

+ 69.0Volts 290.0 121.0 -23.0Volts

+ 69.0Volts 290.0 226.0 -18.5Volts D-4 7-Aminoclonazepam

+ 61.0Volts 287.0 269.0 -12.0Volts

+ 61.0Volts 287.0 241.0 -10.5Volts Oxazepam

+ 70.0Volts 286.0 121.0 -23.0Volts

+ 70.0Volts 286.0 222.0 -18.5Volts 7-Aminoclonazepam

+ 70.0Volts 285.0 154.0 -19.5Volts

+ 70.0Volts 285.0 193.0 -24.0Volts Diazepam

+ 73.0Volts 271.0 165.0 -24.0Volts

+ 73.0Volts 271.0 140.0 -24.0Volts Nordiazepam

CID Energy Capillary Q1 Q3 Collision Analyte

Chromatogram of Benzo Mix,250 ng/mL Calibrator

2.5 5.0 7.5 10.0 12.5minutes

010

MCounts

050

MCounts

050

MCounts

0.0

MCounts

02

MCounts

020

MCounts

04

MCounts

020

MCounts

040

MCounts

0

MCounts

04

MCounts

04

MCounts

271.0>140.0 [-24.0V]

285.0>193.0 [-24.0V]

286.0>222.0 [-18.5V]

287.0>241.0 [-10.5V]

290.0>226.0 [-18.5V]

290.1>198.0 [-24.0V]

292.0>246.0 [-10.5V]

301.0>255.0 [-18.5V]

309.0>281.0 [-23.5V]

314.1>286.0 [-18.5V]

316.0>270.0 [-20.5V]

321.0>275.0 [-18.0V]

Nordiazepam

Diazepam

7-Aminoclonazepam

Oxazepam

D-4 7-Aminoclonazepam

D-5 Diazepam

D-5 Oxazepam

Temazepam

Alprazolam

D-5 Alprazolam

Clonazepam

Lorazepam

Seg 1, Time: 0.05-15.10, Scan Functions: 282031 4107 6182 8257 10333 Scans

Representative Calibration Curves (10-1000ng/mL)7-Aminoclonazepam

Curve Fit: Linear, Origin: Include, Weight: None Resp. Fact. RSD: 8.179%Coeff. Det.(r2): 0.9993y = +1.058993x -0.006779

Peak Size

/ PS

Std.

Amount / Amt. Std. (ng/mL)1 2 3 4 5

0

1

2

3

4

5

Replicates32 2 2 2 2 2

ClonazepamCurve Fit: Linear, Origin: Include, Weight: None Resp. Fact. RSD: 5.639%

Coeff. Det.(r2): 0.9978y = +1.054153x +0.013111

Peak S

ize /

PS Std

.

Amount / Amt. Std. (ng/mL)1 2 3 4 5

0

1

2

3

4

5

Replicates32 2 2 2 2 2

R(2) = 0.999RSD = 8.17

R(2) = 0.997RSD = 5.64

Benzodiazepine Summary

Excellent sensitivity Quantitation limits will be below 10ng/mL for all compounds Excellent quantitation can be achieved using a select few internal standardsAll 8 drugs can be run simultaneously – and many more can be incorporated as to your needsThe 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

Analysis of Amphetamines by LC/MS/MS

Amphetamines Experimental Conditions

Methamphetamine: MS/MS Breakdown Methamphetamine Calibration CurveCalibra tion Cu rve Re por tFile : e :\nor che m \a m ps \a m p h etam in e sd a ta m u lt ip lie r2 .m thD etec to r : 12 00 M as s S pec , Add res s: 4 2

M eth am phe tam ineC urv e Fit : L in e ar, O rig in : Ignor e , W e igh t: N on e R e sp. F a ct . R SD : 5 .31 8%

C o ef f. D e t .( r2 ) : 0 .9 9741 9y = + 0 .031 91 4x +0 .25 72 71

Peak

Size

/ PS

Std.

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

2 50 0 5 0 00 75 00

0

5 0

10 0

15 0

20 0

25 0

30 0

R ep lic ate s 9 3 3 3 3 3

Cal ibra tion Cu rve Re por tFile : e :\no r ch e m \a m p s \a m p h etam in esd a ta m u lt ip lie r2 .m thD ete c to r : 12 0 0 M as s S pe c , Ad d res s: 4 2

M e tham ph eta m in e d 5C urv e Fit : L in ea r, O rig in : Inc lu d e (Ig n or e ), W e igh t : N on e R e sp. F a ct . R SD : 0 .0 00 0 %

C o ef f. D e t .( r2 ) : 1 .00 00 0 0y = + 1 .00 0 0 00 x +0 .0

Peak

Size

Am o un t

0.9 99 9 00 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

2 5

5 0

7 5

10 0

12 5

15 0

M R ep lic ate s 24

Methamphetamine 5-10,000 ng/mL Methamphetamine d5 IS

Chromatographic data of Amphetamines

Spiked: 50 ng/mL in urine

Amphetamine Summary

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

Analysis of Carboxy-THC by LC/MS/MS

Carboxy-THC Experimental Conditions

• 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

• ESI Negative.LC Conditions:

• Column: Varian Pursuit Diphenyl50X2mm, 3μFlow Rate: 0.2 mL/minA: WaterB: Methanol

Time %B0:00 400:30 401:00 953:00 953:01 406:30 40

Injection Volume, 20 μL

• Samples• In a complicated matrix

solution• Standard Solutions prepared

by serial dilutions in DI water

• 50 μL IS added to 1 mL of sample

Chromatograms – Carboxy-THC

Carboxy-THC – 10 ng/mL

Carboxy-THC – 1 ng/mL

Carboxy-THC Calibration Curve Report

Carboxy-THC 1-1000 ng/mL

Carboxy-THC d9 - IS

Carboxy-THC Sample Report Example Carboxy-THC Summary

Excellent sensitivity at the 1 ng/mLDetection limits in the sub ppb levels Linear calibration curve from 1-1000 ng/mLR2 value of 0.998Run time of only 6.5 minutesSample prep does not require derivatization –simply dilute urine in water and inject!