Forensic

Chiral Analysis of Methamphetamine and its Metabolite,Amphetamine, in Urine by CESI-MS
John C. Hudson
SCIEX, Regina, SK, Canada

Introduction
Chiral separation of drug enantiomers is essential in orderto show that the active enantiomer is, in fact, present inforensic specimens. This avoids legal arguments andsimplifies challenges to analytical findings. Chiral analysis ofmethamphetamine (meth) “street” samples yields informationon the clandestine lab synthetic route.1 Chiral analysis is also ofgreat importance in pharma and drug discovery for the detectionof chiral impurities and for quantitative determinations.
In the past, chiral analysis has been done using a combinationof processes, starting with the drug confirmation by hyphenatedmass spectrometry (CE-MS, GC-MS or LC-MS). This was

Figure 1. PFT and counterion flow: (a) rinse with BGE followed by 25 psi/
60 s injection to partially fill the capillary; (b) inject sample (~8.5 nL) with
5 psi for 10 s; (c) voltage separation at 30 kV and (d) separation complete.

followed by separation of the enantiomers and impurities of theCapillary interfaceOptiMS cartridge with 90 cm bare fused-silica
capillary, 150 μm OD, 30 μm ID with
conductive emitter tip

CE instrument

CESI 8000 Plus High Performance
Separation-ESI Module

MS instrument 1

SCIEX TripleTOF® 6600 LC-MS/MS Systemwith Analyst® Software 1.7

MS instrument 2Waters Xevo with MassLynx Software 4.1

ESI voltage

1.25 kV

Sample introduction

Hydrodynamic 5 psi for 10 s

contamination and ion suppression in the electrospray process.

Capillary conditioning

Initial conditioning with MeOH, water, 0.1 M
NaOH, water and BGE

In 2005, Rudaz and Veuthey2 showed that adequate chiral

Background electrolyte(BGE)

25 mM ammonium formate pH 2.85

Separation

25 kV, 277 V/cm, 2.3 μA

Temperatures

Capillary 25 °C; samples 10 °C

drug by a specific chiral separation technique, such as chiralcapillary electrophoresis or chiral chromatography.
Direct connection of chiral separation technology with mass
spectrometry can be problematic. The use of chiral GC andLC columns alone or with mass spectrometry provides,
at best, marginal separation capability. Furthermore, theaddition of neutral or highly sulfated cyclodextrin additives inchromatographic and electro-driven separation modes can cause

separations and identification of enantiomers could be doneusing a sheath-liquid CE-MS technique. Their partial filling
technique (PFT), under countercurrent conditions, employedhighly sulfated cyclodextrin (HSCD) additives to a simple
background electrolyte (BGE) to separate the enantiomers ofamphetamine (amp) derivatives (see Figure 1).
In this work, a low-flow capillary electrophoresis electrosprayinterface for mass spectrometry (CESI-MS) was used with thepartial filling technique, as illustrated in Figure 1, to generate thechiral separation and produce the quantitative data.

Figure 2. CESI 8000 with Opti-MS ® Conditions

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

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Forensic
To 1 ml of urine (or serum, plasma or oral fluid)
1. Add 50 μL of mixed 011 internal standards to 1 mL of urine followed 		by 0.2 mL of cone, NH4OH and vortex.
2. Add 5 mL of 1-chlorobutane and shake for 10 min.
3. Centrifuge at 0 °C for 10 min at 3000 rpm.
4. Evaporate at 40 °C under N2 for 10 min to remove any NH40H,
then add 10 μL of 1% HCI in MeOH. Vortex and continue toevaporate with N2.
5. Add 200 μL of 5 mM BGE to each tube and vortex.
6. Transfer to a 200 μL Microfuge tube (Beckman Coulter).
7. Pressure inject the sample for 10 s at 5 psi.
Figure 3. Liquid-liquid extraction protocol for bio-fluids

Figure 7. Linear regression analysis for amp and meth enantiomers.

Materials and methods
Chemicals: All chemicals were reagent grade and werepurchased online from VWR Int. Highly sulfated-gammacyclodextrin (HS-γ-CD), 20% solution in water, was obtained
from Beckman Coulter Inc. (Brea, CA).
Figure 4. Chiral separation of amp±, meth±, D11-amp± and D11-meth±with a SCIEX TripleTOF® 6600 System and Analyst® Software 1.7.

Drug and metabolite standards: Meth±, amp± and their D11
deuterated internal standards, all at 1 mg/mL were purchasedfrom Cerilliant Corporation (Round Rock, TX). These standard
solutions in methanol were diluted and spiked into volunteer
pooled urine samples. Standard solutions for mass spectrometry
and extractions were prepared at 1 ng/μL in 5 to 50 mM
ammonium formate (pH 2.85).
Urine calibration standards: Urine samples were preparedat 2000 ng/mL. These spiked urine samples were diluted withFigure 5. The OptiMS cartridge from SCIEX provides a sheathless
ESI interface.

blank urine to prepare calibrators from 0.5 to 1000 ng/mL per
enantiomer. The samples were kept at ~4 °C until the time ofanalysis. Spiked urine samples and blanks were prepared byliquid-liquid extraction after the addition of internal standards
at 50 ng/mL of each deuterated enantiomer.
Instrument conditions and extraction protocol: Figure 2and Figure 3 outline CESI-MS parameters and the liquid-liquid
extraction process.

Figure 6. MRM analysis (~20 fg Injected).

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References

Spiked urine samples for meth and amp using D11, deuterated

1. Iwata, U.T.; Inoue, H.; Kuwayama, K.; Kanamori, T.;
Tsujikawa, K.; Miyaguchi, H.; Kishi, T. Forensic applicationof chiral separation of amphetamine-type stimulants toimpurity analysis of seized methamphetamine by capillaryelectrophoresis. Forensic Science International 2006,
161, 92–96.

internal standards (IS) were prepared and analyzed using aliquid-liquid extraction protocol (Figure 3).
The partial filling technique (Figure 1), in which the capillary is
40% filled with BGE containing 0.15% HS-γ-CD, was used toaffect the chiral separation of meth and amp (Figure 4).
The CESI 8000 Plus System with the OptiMS cartridge (Figure5) was used to interface CE and MS, providing the required2. Rudaz, S.; Geiser, L.; Souverain, S.; Prat, J.; Veuthey, J.-L.
Rapid stereoselective separations of amphetamine derivatives
with highly sulfated cyclodextrin. Electrophoresis 2005,
26, 3910–3920.

sensitivity on injections of only 8.5 nL of the extract reconstituted
in 200 μL (~20 fg injected).
Multiple reaction monitoring (MRM) was used for the quantitative
processing (meth: 150.2 → 119.1, amp: 136.2 → 119.2, D11-amp
147.2 → 98.1, D11-meth 161.2 → 97.1).
The chiral CESI-MS separation for the 0.5 ng/mL spiked urineextract is shown in Figure 6. LOD/LOQ was the low calibrator,
0.5 ng/mL of each enantiomer.
For each enantiomer, 10 point calibrations in triplicate over3 orders of magnitude were linear with R2 >0.995 for both methand amp from 0.5 to 1000 ng/mL of urine (Figure 7).

Conclusions
A partial filling technique was adapted to a low-flow capillaryelectrophoresis electrospray interface for mass spectrometry(CESI-MS).
Chiral separation and confirmation of the enantiomers of
methamphetamine and its metabolite, amphetamine, in a single
run, were demonstrated as proof of principle at the sensitivity
that forensic toxicologists require in even the most challenging
case work.

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