Recent Advances in Analysis of Small Molecules for ...c.ymcdn.com/sites/casss.site-ym.com/resource/resmgr/CE_Pharm...for Forensic and Pharmaceutical Scientists ... Waters* Micromass*

Recent Advances in Analysis of Small Molecules

for Forensic and Pharmaceutical Scientists

John C. HudsonBeckman Coulter, Inc.

Fullerton, CA, USA

Presentation Outline

♦ Capillary Zone Electrophoresis

♦ Basic Drug Screening

♦ Confirmation with CE/MS/MS

♦ Chiral Analysis – Crystal Meth

♦ Ion Analysis - GHB







Capillary Zone Electrophoresis

+ +

+

Drug Screening

• Forensic Toxicology

• Drug Development (Clinical Trials)

• Illicit (Street) Drugs

More analyte available

Basic Drug Screening

• Basic drugs in acidic solution are Cations.

H+

CZE + PDA detection = Strength of methodology

• CZE gives better resolution - peak capacity• Voltage injection - cleanup• Process of Elimination with reproducible

mobility data (< 0.3% RSD).• Voltage injection - cleanup• UV Spectra Library with modern search

algorithms.

Injection Methods• Amount of sample injected



mobility data (< 0.3% RSD).• UV Spectra Library with modern search

algorithms.

Mobility with Spectral Confirmation


Spectral Library Search


Spectral Library Search

Trazodoneconfirmed

Mobility• … is the best parameter used for method

validation• … allows superior reproducibility and

accuracy compared to use of migration times.

• … is hardware and capillary independent,• ... allows for easy cross-platform validation

of CE methods.



mobility data (< 0.3% RSD).• UV Spectra Library with modern search

algorithms.

Peak Search using Spectral Library

Mobility

60cm x 50um fused-silica capillary, 100mM phosphate pH 2.380, 25kV, 200nm UV detection







Mobility Search

UV Library Search

Trazodone Detected

Screening Blood for Basic Drugs

Nicotine

Cotinine

IS

ES?Whole Blood

Case Extract

CE/MS/MSCE/MS/MS

Confirmation by CE/MS/MS

Mobility Search

UV Library Search

Trazodone Detected


Nicotine

Cotinine

IS

ES?Whole Blood

Case Extract


CE/MS/MSCE/MS/MS

Mobility Search

UV Library Search

Trazodone Detected


Nicotine

Cotinine

IS

ES?Whole Blood

Case Extract


CE/MS/MSCE/MS/MS

P/ACE™ MDQ System

ESI Interface

Waters* Micromass* Quattro Premier XE* Tandem

Quadrupole Mass Spectrometer

*All trademarks are property of their respective owners.


Minutes

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

µA

0

10

20

30

40

50

60

70

80

90

100PDA - 200nm2001-8669 Exh. 1 Blood CZE 2.38

Cocaine

Cocaethylene

IS

Cotinine

Nicotinamide

2001 Impaired Driving Case

IS

Cocaine MRMs

Cocaethylene MRMs

2001 Impaired Driving Case

2001-8669 cocaine

Time10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00

%

0

100Oct25_06_9 Sm (Mn, 2x3) MRM of 5 Channels ES+

304.2 > 81.87.84e5

Area

12.83140165

14.921044

Cocaine MRM 304.2 81.8

2001 Impaired Driving Case – S/N

Cyclodextrinsand

CE/MS

CE/MS/MS – Calibration Mixturein 1.2% ß-CD CE/MS









Cyclodextrins

• Static Cyclodextrins

• Charged Cyclodextrins

Static Cyclodextrin ( )Environment

+ +

+

Static Cyclodextrin ( )Environment

+ +

+

Cyclodextrins• Static Cyclodextrins

– stationary in the capillary– interact with the enantiomers– form complexes with non-chiral drugs– additional screening information – poor resolutions

2.382.38

1.2 1.2 ßß--CDCDMDAMDMA

MDEA

IS

DPH

Ketamine(K)

Nor-K

Lidocaine

MEGX

Static Cyclodextrins

2.382.38


MDEA

IS

DPH

Ketamine(K)

Nor-K

Lidocaine

MEGX


2.382.38


MDEA

IS

DPH

Ketamine(K)

Nor-K

Lidocaine

MEGX


Cyclodextrins

• Static Cyclodextrins


Charged Cyclodextrins’ Environment

Cyclodextrins• Static Cyclodextrins

– stationary in the capillary– interact with the enantiomers– form complexes with non-chiral drugs– additional screening information – poor resolutions


– highly sulfated, alpha, beta and gamma

– negatively charged – anions– give increased resolution– high resolution -> short-end

injections

O

NH2H3C

CH3OH

H

R-iso mer

HSCD Separations

Rapid Enantiomeric Separation of Basic Drugs and Metabolites:A Universal Strategy for Ultra-Fast Method Development.*

John C. Hudson, Beckman Coulter, Inc., Fullerton, CA, USA

IntroductionIntroduction

This work describes the use of charged cyclodextrins in a continuing search for a universal strategy for separation of enantiomeric drug substances. The use of highly sulfated cyclodextrins (HSCDs), originally proposed by Chapman and Chen (1), is a well established and efficient approach used in many laboratories worldwide. In this study, a group of compounds was selected from a set of drugs and metabolites of pharmaceutical and forensic interest (2). This group of compounds was challenging because it included many closely related metabolites of drug substances in addition to the parentdrugs. The results confirmed those of the earlier study (1) andallowed the current strategy to be used as a model for ultra-fast methods development. The strategy has been applied in the determination of drug purity for the pharmaceutical industry. An example of purity analysis illustrates the steps and practical considerations required for a successful outcome.

Chemicals:Solutions of alpha-, beta- and gamma – HSCD at a concentration of 20% w/v and all other reagents were purchased from Beckman Coulter, Fullerton, CA, USA. The reagents were prepared as per the enclosed product documentation.

Drug and Metabolite Standards:Standards were purchased from Cerilliant Corporation, Round Rock, TX, USA or were obtained as a gift from the Royal Canadian Mounted Police, Forensic Laboratory, Winnipeg, MB, Canada or Dr. Robert Meatherall, St. Boniface Hospital, Winnipeg, MB, Canada.

Solutions of these drug and metabolite standards were purchased or prepared at a concentration of 1 mg/mL and diluted to 25 ppm (25 ng/µL) in distilled and deionized water.

Reference Marker:1,3,6,8-Pyrene tetrasulfonate (PTS), 10 mM in water: 2 µL added to each sample.

Instrument:P/ACETM MDQ Capillary Electrophoresis System (Beckman Coulter, Fullerton, CA) equipped with a Photodiode Array Detector (PDAD) with detection at 200 nm (scanning 190-350) and 32 Karat Version 7 Software.Run Buffers: All chiral separations were performed in 5% HSCD in 25 mM triethylammonium phosphate pH 2.5 unless otherwise noted as either run in 2.5% or 7.5% of the HSCD.

Capillaries and Conditioning:Fused-silica capillaries, 50 µm I.D. x 30 cm (effective length 20 cm) were used in all separations. The columns are rinsed daily with 25 mM lithium acetate containing 0.4% Polyethylene Oxide (PEO,MW 300,000) and 10% ethylene glycol, adjusted to pH 4.75 to speed up column equilibration.

Applied Voltage:The voltage was set at 15 kV (500 V/cm) resulting in running currents of 140 to 180 µamps for 50 µm I.D. columns.

Temperatures:Capillary and sample storage = 22oC

Sample introduction:Pressure injection of 4 s at 0.3 psi or Voltage injection of 10 s at 10 kV.

Material and MethodsMaterial and Methods

ResultsResults1. Of the 692 compounds in a library used in forensic drugs screening (2), 101 were known to be racemic compounds and were used in this study.

2. At a concentration of 5%HSCD, 73 of the compounds were resolved at a resolution of 2 or higher.

3. Twenty-eight (28) of the 101 compounds were selected for additional runs at a concentration of 2.5 and 7.5% of each HSCD to further improve resolution. As a result of these runs, a total of 94% of the 101 compounds were resolved at 2 or greater.

4. Examples of the excellent resolution that can be achieved are shown in Figures 1 and 2.

5. This strategy was applied to a very complex racemic drug containing four chiral centers. Prior to this, the existing method used a chromatographic approach where the enantiomers were poorly resolved. To fully resolve the Active Pharmaceutical Ingredient (API) and 3 enantiomers, this systematic process was applied as follows and is illustrated in Figure 3.

Refer to steps A to E in Figure 3 (right):

A. Pressure injection of the three enantiomers) at 10% of the API run in each HSCD at 5% was performed.

B. The best resolution between 5% systems, was obtained in Beta-HSCD. Improved resolution was obtained in 7.5% Beta-HSCD.

C. The capillary length was increased from 20 to 30 cm to the detector (40 cm total).

D. Voltage injection was necessary for increased sensitivity andresolution.

E. Low level purity determination result was made possible withHSCDs and voltage injection.

Method development was completed in less than 3 days.

Table 1: Resolution and HSCD Sytems for 101 Basic Drugs and Metabolites

Drug or Metabolite Resolution System* Drug or Metabolite Resolution System*

Acebutolol 2.4 Gamma Fluoxetine, Nor- 9.8 GammaAdrenaline, Nor- (Norepinephrine) 3.5 Gamma 2.5 Glutethimide 11.8 GammaAminorex, Cis-4-methyl- 4.5 Gamma Ketamine 2.2 GammaAmphetamine 33.2 Gamma Ketamine, Nor- 4.5 AlphaAmphetamine, 2,3-Dimethoxy- 16.6 Gamma Labetolol 5.1/2.9/2.1*** Gamma 2.5Amphetamine, 2,4-Dimethoxy- 22.5 Gamma MBDB 7.5 GammaAmphetamine, 2,5-Dimethoxy- 17.2 Gamma MDA, 2,3- 16.7 GammaAmphetamine, 2,5-Dimethoxy-4-bromo- 9.9 Beta MDA, 3,4- 14.6 GammaAmphetamine, 2,5-Dimethoxy-4-ethyl- 11.6 Gamma MDEA, 3,4- 7.1 GammaAmphetamine, 2,5-Dimethoxy-4-methyl- 13.8 Gamma MDMA, 2,3- 9.7 GammaAmphetamine, 2,5-Dimethoxy-4-propyl- 6.0 Gamma MDMA, 3,4- 7.8 GammaAmphetamine, 2,6-Dimethoxy- 4.5 Gamma Methadone 26.6 BetaAmphetamine, 3,4-Dimethoxy- 14.4 Gamma Methamphetamine 11.8 GammaAmphetamine, 3,5-Dimethoxy- 4.6 Gamma Methoxamine 44.4 GammaAmphetamine, 3-Methoxy-4,5-methylenedioxy- 4.6 Gamma Methylphenidate 14.5 GammaAmphetamine, N-Ethyl- 14.7 Gamma Metoprolol 4.2 Alpha 2.5Amphetamine, 4-Methylthio- (4-MTA) 13.4 Gamma Mexilitine 2.7 Gamma 7.5Amphetamine, Hydroxy- 30.6 Gamma Midazolam 3.1 GammaAtenolol 3.4 Gamma Mirtazapine 5.4 GammaBisoprolol 2.2 Gamma 2.5 Mirtazepine, N-Desmethyl 7.5 GammaBrompheniramine, Dinor- 0.8 Beta 7.5 Nadolol 2.6/1.3/split*** GammaBrompheniramine, Nor- 1.0 Beta Nefopam 7.6 AlphaBupropion 10.1 Alpha Oxprenolol 10.0 BetaBupropion, Erythroamino- 7.5 Gamma Pentazocine 7.2 Gamma 7.5Bupropion, Hydroxy- 6.3 Beta Pheniramine 2.8 Beta 7.5Bupropion, Threoamino- 7.6 Alpha Phenmetrazine 19.5 AlphaButriptyline, N-desmethyl- 1.1 Alpha Phenylephrine, N-Desmethyl- 3.6 AlphaChloroquine, N,N,-Didesethyl- 1.9 Gamma 7.5 Phenylpropanolamine 42.6 GammaChloroquine, N-Desethyl- 1.1 Alpha Pindolol 2.0 Beta 7.5Chlorpheniramine 2.5 Beta 7.5 PMA (p-Methoxyamphetamine) 25.3 GammaChlorpheniramine, Dinor- 1.0 Beta 7.5 PMMA (p-Methoxymethamphetamine) 13.1 GammaChlorpheniramine, Nor- 1.1 Beta 7.5 Propranolol 4.3 Alpha 2.5Citalopram 11.1 Gamma Pronethalol 2.5 Alpha 2.5Citalopram N-Oxide 11.0 Gamma Propoxyphene 7.3 AlphaCitalopram, Dinor- 8.0 Gamma Pseudoephedrine 5.2 GammaCitalopram, Nor- 8.0 Gamma Quetiapine 4.4 GammaCyclazocine 3.8 Beta Quinidine 2.3 AlphaCyclobenzaprine 1.9 Gamma 7.5 Salbutamol 8.3 BetaCyclobenzaprine, N-Desmethyl- 5.4 Alpha Tetramisole 3.6 GammaDesloratadine 6.4 Gamma Tramadol 13.3 GammaDihydro-N,N-dimethyl-5-methylene- ** 3.2 Alpha Tramadol, Nor- 10.3 GammaDisopyramide, p-Cl 4.7 Beta Trimipramine 7.3 AlphaDisopyramide, N-Dealkylated- 4.6 Alpha Trimipramine, Nor- 2.0 AlphaDoxapram 7.2 Gamma Venlafaxine 5.5 GammaDoxylamine 2.2 Gamma 7.5 Venlafaxine, O-Desmethyl- 4.3 GammaEDDP (Methadone Mtb.) 3.0 Beta 7.5 Verapamil 7.1 AlphaEMDP (Methadone Mtb.) 7.5 Gamma Verapamil, Nor- 6.9 AlphaEphedrine 6.1 Alpha Zopiclone 24.0 GammaEphedrine, Hydroxy 15.6 Gamma Zopiclone N-Oxide 23.6 GammaEsmolol 3.1 Gamma Zopiclone, Nor- 31.6 GammaFluoxetine 12.0 Alpha 2.5

* All systems 5% unless indicated** Dihydro-N,N-dimethyl-5-methylene-5H-dibenzocycloheptene-10-ethanamine, 10,11-*** Compound with 2 chiral centers

ConclusionConclusionFinding a universal method for chiral separations has been a challenge for many years. An important group of drugs and metabolites, expected to be frequently detected because of widespread use by the general population, was used to evaluate the proposed universal strategy. These 101 racemic drugs and metabolites are of interest to both the pharmaceutical and forensic communities. The compounds were rapidly screened and resolved with resolutions of 2 or greater for over 94% of the group.

ReferencesReferences

1. Chapman, J.D and A-F. Chen, LCGC Europe, January 33 - 37 (2001)

2. Hudson, J.C. et al., Can. Soc. Forens. Sci., 31(1) 1 - 29 (1998)

.

Minutes1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

µA

-160

-140

-120

-100

-80

-60

-40

-20

0

O-Desmethylvenlafaxine Venlafaxine

PTS

Figure 1: Methoxamine – Mobility Marker and Internal Standard Figure 2: Venlafaxine and Metabolite in 5% Gamma-HSCD

Figure 3: Rapid Method Development for Purity Analysis Using Highly Sulfated Cyclodextrins

* Poster Presented at: CE in the Biotechnology & Pharmaceuticals Industries 10th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules October 12-16, 2008 For Research Use Only; Not for use in diagnostic procedures.

A

B C

ED

Dilution 20/1 of

the 10%Solution

PDA Detection of Enantiomers & API

Cyclodextrins

• Rapid Method Development

• Roche Example

Roche – New Drug Substance• 4 Chiral Centers.• 4 Enantiomeric Standards Available.• SSSS Configuration is Active Pharmaceutical

Ingredient (API).• Major Enantiomer – RRRR.• Two other impurities – RSSS and SRRR.• Molecular Weight 377 as free base.• Available as HCl salt – very water soluble.• All injections from water.

Minutes1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

µA

-200

-175

-150

-125

-100

-75

-50

-25

0

Beta-HSCD 5%

PTS

Beta – Best HSCD Candidate

Minutes2.00 2.02 2.04 2.06 2.08 2.10 2.12 2.14 2.16 2.18 2.20 2.22 2.24 2.26 2.28 2.30

AU

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

µA

-215

-210

-205

-200

-195

-190

-185

-180

-175

-170

RRRR

SSSS

SRRR

RSSSBeta-HSCD 5%

Beta – Best HSCD Candidate

Minutes1.975 2.000 2.025 2.050 2.075 2.100 2.125 2.150 2.175 2.200 2.225 2.250 2.275 2.300 2.325 2.350 2.375 2.400

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

µA

-210

-200

-190

-180

-170

-160

-150

RRRR

SSSS

SRRRRSSS

Beta-HSCD 5%

Mixture of the 4 enantiomers

Mixture – Pressure Injection

Minutes6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6

AU

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

µA

-140

-120

-100

-80

-60

-40

RRRR

SSSS

SRRRRSSS

Column:

50 µm i.d.

30 cm to det

40 cm total

Beta-HSCD 7.5%

Injections:

Pressure

0.3 psi

4 sec.

Mixture in 7.5% Beta-HSCD

Minutes1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

µA

-225

-200

-175

-150

-125

-100

-75

-50

-25

0

25

Voltage Injection:“Reversed

Polarity”

Voltage

10 kV

10 sec.

Enantiomers

Mixture – Voltage Injection

Minutes1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

µA

-225

-200

-175

-150

-125

-100

-75

-50

-25

0

25

Voltage Injection:“Reversed

Polarity”

Voltage

10 kV

10 sec.

Enantiomers

Needs dilution

20/1

PDA Shows Mixture Components

After Dilution – Sharp Peaks

Voltage

Injection:“Reversed

Polarity”

Voltage

10 kV

10 sec.

Dilution 20/1

of the 10% solution

Minutes6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2

AU

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

µA

-225

-200

-175

-150

-125

-100

-75

-50

-25

0

25

Dilution 10/1

of the 1% solution

Dilution - 0.1% Purity Possible

RRRRSRRR

RSSS

SSSSRange 0.12 to 0.17%

Purity Study – 0.1% Results

Methamphetamine (Crystal Meth)• Methamphetamine and its metabolite,

amphetamine

• HSCD discriminating power

• Migration time reversals

• Fast Method Development

Meth(M), Amp(A) and IS in Alpha 5%

M+M-

A-A+

IS

Meth/Amp/IS in Alpha or Beta 5%

M+ M-

A-A+

IS

M+M-

A-A+

IS

Meth/Amp/IS in Alpha or Beta 5%

M+ M-

A-A+

IS

M+M-

A-A+

IS

Meth/Amp/IS in Alpha, Beta & Gamma 5%

M+ M-

A-A+

IS

M+M-

A-A+

IS

M+ M- A-A+

IS


M+ M-

A-A+

IS

M+M-

A-A+

IS

M+ M- A-A+

IS


M+ M-

A-A+

M+M-

A-A+

M+ M- A-A+

Methamphetamine

Amphetamine

IS

M+ M-

A-

A+

IS

Normal

Fast

Fast Method Development - Amp/Meth

Forensic Applications Ion Analysis

• Clandestine Labs - Chemicals• Poisoning cases• Tampering cases – HCl in juice• KCl in syringe• Environmental Cases• DFSA (GHB)

Cations Test Mixture(Designed for Acidic Drug Counterions)

1

2

3

4

5

6

1 = Chloride

2 = Nitrate

3 = Sulfate

4 = Azide

5 = Fluoride

6 = Phosphate

Inorganic Anions Test Mixture

Organic Anions Test Mixture

Organic Anions Test Mixture

GHB

GHB Standard 100 ng/µL in Water

GHB IS

HOCH2CH2CH2COO-

GHB in Extracts of 0.1 mL of Blood

GHB100

10

IS

Blank

Test MixButryate

GHB in Extracts of 0.1 mL of Blood

GHB100

10

IS

Blank

Test MixButryate

AcknowledgementsRCMP Forensic Laboratory – Winnipeg, Canada

Cam LyttleVal Mason-Daniel (Retired)Shirley Treacy

Hoffman –La Roche AG, Basil, SwitzerlandMarco GirodMaurice Notter

Beckman Coulter, Fullerton CAJeff Chapman

Hans Dewald

For Research Use Only; not for use in diagnostic procedures

Recent Advances in Analysis of Small Molecules

for Forensic and Pharmaceutical Scientists

John C. HudsonBeckman Coulter, Inc.

Fullerton, CA, USA

Documents

Recent Advances in Analysis of Small Molecules for ...c.ymcdn.com/sites/casss.site-ym.com/resource/resmgr/CE_Pharm...for Forensic and Pharmaceutical Scientists ... Waters* Micromass*