Sheathless Capillary Electrophoresis - Mass Spectrometry:
The High Sensitivity Porous Sprayer (HSPS) for the Forensic Analysis
of Drugs and Metabolites
John C. Hudson, Beckman Coulter, Inc.
Discovery Solutions Business Center, Brea, CA, USA

Introduction
In Forensic Drug Analysis there is always a need for increased
sensitivity to provide better detection in challenging casework.
Samples may only be obtained hours or days after the crime has
been committed and the drugs may have been metabolized or themetabolites excreted. In this work we apply a prototype sheathlessinterface for Capillary Electrophoresis-ESI-Mass Spectrometry (CEMS) to the objective of routine screening for drugs and metabolites at
sub-therapeutic levels in biofluids. Levels of detection (LOD) for acustom designed Test Mixture were determined for 15 drugs andillustrated for one well known drug, diphenhydramine (DPH).
Extracts of spiked human urine were analyzed and the LOD wasdetermined for each of the 15 drugs. The ruggedness of the HSPS
interface was tested in automated batch analysis of the spiked urine
extracts and further in Case Studies on the antiemtic drugs,
metoclopramide, and in an exploration of the urine metabolic profile
after ingestion and metabolism of the antimalarial drug, chloroquine.

Improvements In:• Sensitivity• Resolution• Robustness• Reduction of artifacts
Exclusively Licensed from UT-Austin

Figure 2: Schematic of the High Sensitivity Porous Sprayer (HSPS). The
electrical connection is made from needle to conductive liquid throughthe porous sprayer tip to the BGE.

Figure 3: LOD/LOQ for Serial Dilutions of DPH in Test Mixture show excellent linearity
and a S/N of 824 for 1 ng/mL.

Material and Methods
Chemicals:
All chemicals were Reagent Grade and were purchased from VWR
International.
Drug and Metabolite Standards:
Standards were purchased from Cerilliant Corporation, Round
Rock, TX, USA. Stock solutions prepared at a concentration of 1
mg/mL and were further diluted to 5, 1 or 0.1 ng/µL in 5 mM
Ammonium Formate (pH 2.85).
Operating Conditions:
Capillary

Temperatures
Background
Electrolyte (BGE)

90 cm bare-fused silica,150µm o.d., 30µm i.d., at 200-300 V/cm, 2.4 to 2.8µamps
Capillary and sample storage = 25 oC
50 mM Ammonium Formate, pH 2.85

SampleHydrodynamic 16s at 34.5 mBar orIntroduction
Electrokinetic 10kV for 16s
CE Instrument
Beckman Coulter PA 800 Enhanced
MS Instrument
Waters Xevo TQ with MassLynx 4.1
Sheathless
High Sensitivity Porous Sprayer (HSPS)
Interface
Prototype (Beckman Coulter, Brea, CA).
Conductive Liquid 0.7% Formic Acid
ESI Voltage
1.2 to 1.8 kV
HSPS
The capillaries were initially conditionedConditioning
with MeOH, water, 1N NaOH, water andBGE.

Results
1. A Drug and Metabolite Test Mixture utilizing Multiple Reaction Monitoring(MRM) of 15 drugs and an internal standard on a Tandem Quadrapole MS
was used in the evaluation (Figure 1).
2. A sheathless prototype HSPS (Figure 2) was used to interface CE andESI-MS.
3. Linearity and LOD determinations were done using serial dilutions ofaqueous solutions of the Test Mixture. The LOD values for all components
were found to be less than 1 ng/mL. See Figure 3 for an example
component from the Test Mixture, diphenhydramine (DPH) .
4. Urine samples were spiked with the Test Mixture and internal standard,
doxapram, and were processed with a standard liquid-liquid extraction
protocol (1).
5. As an illustration of the required detection sensitivity in Forensic Drug
Analysis, the common drug, diphenhydramine, was selected from the urine
analysis results. Levels after therapeutic dosing of the drug typically resultin levels of 20 ng/mL in blood (2) and are usually an order of magnitude
higher in urine (Figure 4).

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Figure 4: LOD Requirements for Blood or Urine Analysis are based on TherapeuticLevels in Blood. Detection at sub-therapeutic levels is necessary in difficult
cases such as Sexual Assault and Impaired Driving scenarios.

6. Detection at lower levels (sub-therapeutic) is important in cases where asignificant period of time has elapsed after intake of the drug and collectionof the samples, e.g. sexual assaults, impaired driving.

510111

489,1213141615

Figure 1: Drug/Metabolites Test Mixture and MRM Conditions

2. CQ, N-Desethyl-hydroxy308.2

5. CQ, N-Desethyl- 294.2
Figure7: N,N-DidesethylCase Study 2: Chloroquineand Metabolites determined over a three6. CQ,
264.3
day period exhibit similar metabolic profiles with the largest component
free parent drug, chloroquine and four other metabolites.

Conclusions

7. Diphenhydramine (DPH) was easily detected at an LOD and LOQ of
0.25 ng/mL of urine. This is a factor of 80 times lower than the required
detection level of 20 ng/mL after therapeutic administration (Figure 5).

A High Sensitivity Porous Sprayer (HSPS) has been integrated into asheathless interface for CE-ESI-MS. The HSPS provides superior transfer ofanalytes to the mass spectrometer and maintains the intrinsic attributes ofcapillary electrophoresis such as resolution. Routine detection of common
drugs in biological fluids for forensic laboratories is facilitated by thisadvancement and can be applied to the most challening of forensic drug cases.
The process is robust and automated and should be of great value to analyticallaboratories that work with charged analytes.

8. The HSPS interface was further tested for ruggedness on positive urine
samples provided by volunteers in two case studies.

10. Case Study 2 illustrates the resolution of chloroquine and metabolites
provided by CE and maintenance of this resolution by the HSPS interface(Figure 7). Chloroquine and four of its metabolites were detected in all
samples over a four day collection period. This confirmed that the parent
drug was excreted unchanged and in high amounts, thus providing
protection against malaria in a travel dosing scenario.

1. Doxapram IS 379.5

4. CQ 320.3

References

72

CQ, Metabolites, IS (M+1)

3. CQ, Hydroxy- 336.4

9. In Case Study 1 (Figure 6), urine was provided by a volunteer after aminor surgical procedure. The antiemetic drug, metoclopramide, waseasily detected and confirmed (3 ions provided by 2 MRMs) after
therapeutic administration. The results were obtained in automated runs ofurine extracts without any matrix related problems.

3Figure 6: Case Study 1: Metoclopramide was confirmed in a Patient’s Urine using thewidely accepted criteria of the presence of three ions including the M+1 andthe two most prominent daughter ions.

1. Hudson, J.C. et al., Can. Soc. Forens. Sci., 31(1) 1 - 29 (1998).
2. Baselt, R.C. Disposition of Toxic Drugs and Chemicals in Man, pp. 489-492, 8thEdition, Biomedical Publications, Foster City, California, 94404 (2008).
Figure 5: The LOD/LOQ for Diphenhydramine in Urine determined using spiked urine
samples allows detection easily in the most challening cases.

For Research Use Only; prototype not for use in diagnostic procedures.