CESI-MS Analysis of Biofluids of Basic Drugs
and Metabolites

Blood Banking
Capillary Electrophoresis

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

Centrifugation
Flow Cytometry
Genomics
Lab Automation
Lab Tools

Introduction
Capillary Electrophoresis (CE) has been used for over
15 years to screen biofluids, which include whole
blood, serum, plasma and urine.(1-4) Confirmation andquantification of the presence of a drug or metabolite
detected by CE has traditionally been done independently
after the screening process by gas, liquid chromatography
or sheathflow CE(5) coupled to Mass Spectrometry (MS).
In this paper, the successful use of an integrated CE and
ESI process known as CESI, is described in the context
of the analysis of biofluids for drugs and their metabolites.
The sensitivity is greatly improved by the use of this very
low flow process. This hyphenated technique can provide
screening, confirmation and quantification of drugs
and metabolites in a single process with detection at
0.25 ng/mL or lower in biofluid.

Experimental
Chemicals and standards
Drug and metabolite standards were obtained from
Cerilliant Corporation, Round Rock, TX, USA; SigmaAldrich, St. Louis, MO, USA; and USP, 12601
Twinbrook Parkway, Rockville, MD, USA. All standards
were dissolved in 5 mM ammonium formate at pH
2.85 buffer at the concentration indicated in Table 1.
Quantitative analysis used a spiking mixture at 3 times
the concentration shown in Table 1 in each milliliter ofbiofluid. Doxapram was used as Internal Standard (IS),and it was added separately at 50 nanograms per mL ofbiofluid.
Reagents
Distilled and deionized (DDI) water was obtained froma Barnstead Nanopure Infinity ultrapure water system.
Rinse solutions of methanol, 1N sodium hydroxide and or
0.1N hydrochloric acid were all reagent grade and wereobtained from VWR Scientific, Bridgeport, NJ, USA. The
Background Electrolyte (BGE) and Conductive Liquid(CL) solutions were prepared as follows: BGE (50 mM
ammonium formate, Alfa Aesar P/N A10699 at pH 2.85)
was prepared by diluting 962.5 microliters of formic acid
IB-15841A

Particle Characterization(98%, EMD P/N FX0440-5) with DDI
water to a final volume of 500 mL, andthen adjusting its pH to 2.85 with a
50 mM ammonium formate solution(0.813 g ammonium formate dissolved in
250 mL of DDI water). CL (0.7% formic acid)was prepared by diluting 714 microliters of formic acid(98% purity) with DDI water to a final volume of 100 mL.
Alternatively, the BGE can be used as the conductive
liquid. Both BGE and CL solutions were stored at 2°C
to 8°C when not in use. Both solutions were brought toambient temperature and sonnicated before use. The
BGE was filtered through a 0.2 µm membrane filter justprior to use.

#

Compound

Conc.
ng/µL

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Nicotine
Pheniramine
Chlorpheniramine
Tryptamine
Cotininem-CPP
Methoxamine
Diphenhydramine
Lidocaine
Hydroxyzine
Metoprolol
Trazodone
Haloperidol
Doxapram IS
Verapamil
Loperamide

3
1
1
1
3
1
2
1
1
1
1
1
1
1
1
1

SourceCer-Cerilliant
S – Sigma-Alrich
USP – United States
Pharmacopeia
N-008 Cer
P-045 Cer
C-036 Cer
246557 S
C-016 Cer
C-089 Cer
M 6524 S
D-015 Cer
L-018 Cer
H8885 S
M-123 Cer
T-030 Cer
H-030 Cer
USP 1225000
V-002 Cer
L 4762 S

Table 1: Drug and metabolite performance standard withsource information.

Figure 1. Schematic of the OptiMS capillary.

with a capillary voltage range from 1200 to 1400V. MS/
MS was performed using Multiple Reaction Monitoring(MRM) (Table 2) with argon as the collision gas at
0.15 mL/min.

Instrument conﬁgurations
CE instrument
All experiments were performed using a prototype CESI
8000 High Performance Separation – ESI Module*(Beckman Coulter, Inc., Brea, CA, USA). Data wereacquired and the instrument was controlled by CESI
8000 Software. Prototype OptiMS Silica Surface
Capillary Cartridges (150 μm OD, 30 μm ID, 90 cm long)with a porous tip were obtained from Beckman Coulter.
All separations were performed at a capillary temperatureof 20°C. The system sample storage temperature was set
to a temperature of 10°C. Only applied voltage and CE
current data were acquired on the CE.

OptiMS capillary
The OptiMS capillary (Figure 1) used in this workfeatures a porous tip, which was created from a
150 μm OD, 30 μm ID capillary with the polyimide
removed from the tip area. This combined ESI source,
interface and separation capillary, are enclosed in a
cartridge with a stainless steel needle in a retractable
tip assembly (Figure 2). Electrical contact was madebetween the background electrolyte in the capillarythrough the porous section of the capillary tip andﬁnally through a conductive liquid which ﬁlls the space
between the needle and porous tip. The conductive
liquid is supplied using pressure rinsing from the outlet
side of the CESI instrument.

MS instrument
MS data were acquired on a Waters Xevo tandem mass
spectrometer (Waters Corporation, Milford, MA, USA).
The instrument was operated in ESI positive ion mode2#Compound

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Nicotine
Pheniramine
Chlorpheniramine
Tryptamine
Cotininem-CPP
Methoxamine
Diphenhydramine
Lidocaine
Hydroxyzine
Metoprolol
Trazodone
Haloperidol
Doxapram
Verapamil
Loperamide

Precursor ion
(m/z)
163.2
241.2
275.3
161.2
177.2
197.2
212.4
256.3
235.3
375.3
268.2
372.3
376.3
379.4
455.8
477.5

Product ion
(m/z)
130.1
196.3
230.1
144.2
80.1
154.1
194.4
167.2
86.1
201.2
73.7
176.2
165.2
292.3
165.4
266.2

Cone voltage
(V)
30
25
20
17
30
25
25
15
25
25
25
20
25
25
30
30

Collision energy
(eV)
22
18
19
13
25
20
10
13
20
20
25
27
25
22
30
27

Table 2: MRM transitions and conditions for the performance standard.

Figure 2: Schematic of the OptiMS Cartridge and retractable tip assembly. The data was generated on a prototype similar
to the one shown above.

3

sample from one run to the next. The conductive liquid(CL) supply to the needle surrounding the porous tip wasreplenished prior to each run (line 3 of Figure 4) from theoutlet side of the CE instrument by a reverse pressure
rinse. The sample was electrokinetically injected at the
inlet side using a 10kV voltage injection of 16 seconds.
The sample injection was followed by a short 0.5 psipressure injection of BGE for 10 seconds to ensure
good quantitative reproducibility of injections. During all
steps in the separation method, the conductive liquid
capillary installed in the outlet side of the instrument was
positioned in BO:A1. The vials were incremented every
12 runs to prevent carryover and buffer depletion effects.

Preparation of the OptiMS silica
surface capillary
CE conditioning method
New OptiMS capillaries were conditioned by rinsing,in sequence, with methanol to remove organic material
from capillary manufacturing, DDI water to remove themethanol, 1N NaOH to ﬁll the capillary and condition
its surface during a 60 min. wait period. DDI water wasused to remove NaOH and, ﬁnally, a long rinse withbackground electrolyte (BGE) was used to condition
the capillary surface (Figure 3). This conditioning method
was performed with the porous sprayer retractable
tip, which was immersed in DDI water. Note: Thisconditioning method is used once on a new sprayer.
However, this same method can be repeated asnecessary as a cleaning procedure or to improveseparation performance.

Shutdown methods
Following the completion of a sequence of runs, thecapillary was preserved for future use by different
methods, depending on the situation.

CE separation method
The sample components were separated by CE using
the following method (Figure 4). First, the capillary wasrinsed twice with BGE rinses, a three-minute rinse
followed by a ﬁve-minute rinse, both at 50 psi. This
double rinse ensured that there was no carryover of

Short-term storage (<24 hrs.)
The housing containing the sprayer tip was immediately
removed from the system and submerged in DDI water
overnight to be used the next day.

Figure 3: Time program for new capillary conditioning methods.

Figure 4: Time program for separation method.

4

Long-term storage (>24 hrs.)
The separation capillary was rinsed sequentially with
DDI water and methanol, followed by the application ofvoltage across the capillary ﬁlled with methanol to remove
any ionic material that might be present in the porous
sprayer tip (Figure 5). Then, the separation capillary wasdried by ﬂushing with air. The CL capillary was rinsed
with water, methanol and air. The retractable housing
end was sealed with Paraﬁlm and the whole assembly
was stored at 2°C to 8°C.

Unattended shutdown
Use the time program below (Figure 6) to decrease therinse ﬂow, thus preventing the capillary from drying out
while maintaining the current MS operating conditions.
Buffer tray conﬁguration
Vials containing 1.5 mL of reagents were placed in
the buffer trays prior to the use of the conditioning,
separation or shutdown methods (Figure 7). All vials
were capped with blue caps. An empty capped vial
was used to rinse capillaries with air.

Figure 5: Time program for storage method.

Figure 6: Time program for unattended shutdown method.

Inlet Tray

Outlet Tray

Figure 7: Buffer tray vial conﬁguration for all methods.

5Use an infusion standard to optimize the location of thesprayer tip relative to the entrance of the MS source. The
optimal X-Y-Z sprayer position will generate the highest
signal response from the mass spectrometer.

Evaluating system performance by runningthe performance standard test mix
An assessment of the performance of the CESI-MS
system must be made before running any samples. In
this work, the separation was optimized for all 16 drugs
and metabolites covering the useful mobility rangeof compounds with molecular weights under 525 Da(Figures 8 and 9).

Step 3. Test the CESI-MS system for performance
Access the sprayer performance by monitoring the CE
electrical current generated during the separation, theminimum ESI voltage required to establish a stable spray,
the results from the infusion standard X-Y-Z positioning
and the MS data from a performance standard. The
performance standard described in this article isrecommended as the separation has been optimized
for drug and metabolite screening. This mixture should
be run daily and the results stored to form the basis forlong-term comparison of the performance of individualinterfaces and the overall system. See “System
Performance” and Figures 8 and 9.

Running the CESI-MS System
In order to ensure that high quality data are acquired, all
the components of the CESI-MS system must be working
properly. Each component should be checked separately
and then optimized as an integrated system. Follow thesesix steps to ensure that high performance is achieved.
Step 1. Qualify and calibrate the instruments
Both the CESI 8000 High Performance Separation –ESI Module and the MS instrument must be maintained
and calibrated prior to integrating them via CESI. This
process requires testing each instrument independently
to ensure that they are operating within manufacturers’
speciﬁcations.

Step 4. Prepare samples from the bioﬂuid
Best results are obtained from an extract of the bioﬂuid
using a preconcentration protocol. Methods such as
liquid-liquid extraction (LLE), and solid-phase extraction(SPE) provide suitable extracts for CESI-MS. Established
LLE protocol(3, 8) using urine is outlined in Table 3. This
procedure can also be used for whole blood, plasma andserum.

Step 2. Install and prepare the interface
Install the previously-conditioned OptiMS Capillary.

Figure 8: Total Ion Chromatogram (TIC) of the performance standard test mix.

6

Figure 9: MRM chromatograms for the performance standard of 16 compounds.

7

Urine**Concentration
100
50
25
10
5
1
0.5
0.25
Blank + IS
Blank

Volume ofStandard Sol’n
20 μL
10 μL
5 μL
10 μL
5 μL
10 μL
5 μL
2.5 μL
N/A
N/A

Conc. ofStandard Sol’n
5 ng/μL
5 ng/μL
5 ng/μL
1 ng/μL
1 ng/μL
0.1 ng/μL
0.1 ng/μL
0.1 ng/μL
N/A
N/A

Volume of IS
Sol’n
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
10 μL
N/A

Conc. of InternalStandard Sol’n
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
5 ng/μL
N/A*See Standards and Table 1. **In duplicate.

Table 3: Design of Experiment (DOE) for spiked urine samples.

8. Transfer the sample solution to 200 μL PCR vials;
cap and centrifuge at 14,000 rpm for 20 minutes toprecipitate any particulate material.
9. Position the samples in the CESI 8000 inlet sample
tray and inject using an applied voltage of 10 kV for
16s followed by a short BGE pressure injection for
10s at 0.5psi.

ProtocolQuantitative Analysis of Bioﬂuids – An Example
Using Spiked Urine Samples.*Drug-free urine samples (1 mL each) were spiked withthe performance standard to yield concentrations ranging
from 0.25 to 250 ng/mL of urine. Doxapram InternalStandard (IS, 50 ng/mL) was added to each tube,
except the Blank (Table 3).

Step 5. Prepare data acquisition control sequences
The CESI 8000 provides the sample automationneeded to collect a series of runs. The sequence(Figure 10) uses a method for voltage injection from theperformance standard and reconstituted urine samples.
A corresponding sequence or sample table from theMS software must be synchronized with the sequenceof the CESI 8000 (a portion of the table is shown in
Figure 11). The MS sample table controls the operationof the applied ESI voltage – turning it on while rinsing theinterface, off while injecting using the CE voltage, andback on when collecting acquisition data.

Extraction Procedure for Bioﬂuids
The extraction of the drugs and metabolites from urine
was done using a LLE method.(3) The procedure involved
the following steps:
1. Add an internal standard, (Doxapram, 50 ng) to
1 mL urine aliquots in 15 mL screw-capped tubes
and vortex for 5 seconds.
2. Spike standards into 1 mL of drug-free urine samples
for use as calibrators and vortex.
3. Add 200 μL of concentrated ammonium hydroxide(28-30%, EMD P/N AX1303-6) to each tube followedby vortexing.
4. Add 5 mL of 1-Chlorobutane to each tube. Cap thetubes and shake on a ﬂat-bed, rotary shaker or similar
device for 10 minutes.
5. Centrifuge the samples at 1500 rpm (Beckman
Coulter Allegra X-22R equipped with SX4250 rotor)
for 10 minutes under refrigeration (0°C – 4°C).
6. Decant the organic layer into 12x75 mm culture
tubes and evaporate under nitrogen (or other suitableevaporation system) just to dryness. Do not overevaporate because it will result in the loss of volatiledrugs (e.g. nicotine).
7. Dissolve the residue in 100 μL of 5 mM ammonium
formate, pH 2.85 by vortexing followed by centrifuging
the 12x75 tubes at 1200 rpm for 2 minutes.

Step 6. Data processing
Data collected by the mass spectrometer software canbe manually processed or, preferably, automatically
via modiﬁcations to the sample table after the full dataacquisition is complete. Lines in the sample table which
are only used to control the ESI voltage are removed andan example is shown in Figure 12.
Step 7. Interpretation of results
In this work, the results for the quantitative urine extracts
provide assessment parameters such as linearity and limit
of detection (LOD).

8

Results
1. Sample acquisition was controlled by the CESI 8000 sequence (Figure 10).

Figure 10: CESI 8000 sequence for urine calibration.
2. MS data were acquired using the MassLynx sample table, a part of which is shown in Figure 13.

Figure 11: MassLynx sample table for control of the ESI voltage and data acquisition.
Sample data ﬁles are acquired in sets of three as illustrated in Figure 11. The ﬁrst ﬁle is used to keep the ESI voltage
on during rinsing the capillary. The second ﬁle is used to turn the ESI voltage off during injection and the following BGE
injection. The third ﬁle in a set of three is the collection of the actual sample data.

9

After data acquisition was complete, the data were processed using the sample table is shown in Figure 12. Lines in theoriginal sample table which control the ESI voltage were removed before processing.

Figure 12: MassLynx sample table for data processing.
3. Linearity was assessed for the 15 drugs and metabolites using Peak Area Ratios (PAR) to the internal standard,
doxapram.

10

4. As examples, the Total Ion Chromatogram of the urine extract and the MRMs for four representative compounds,
pheniramine, m-CPP, diphenhydramine and loperamide are shown below:

Figure 13: TIC and MRMS for four representative drugs and metabolites.

Calibration results are summarized in Table 4. Methoxamine and metoprolol were excluded from the table as they arepoorly ionized in positive ESI MS. Tryptamine was also excluded as it is an endogenous compound always present in urine.
It is important not to over evaporate the residues because nicotine and cotinine may be lost with prolonged drying. The
observed limit of detection was 0.25 ng/mL of urine (the low calibrator) for the drugs and metabolites quantitated. This limit
of detection is well below sub-therapeutic levels and will provide the detection limits needed for the most challenging of
forensic case work.
CompoundNicotine
Pheniramine
Chlorpheniramine
Cotinine
m-CPP
Diphenhydramine
Lidocaine
Hydroxyzine
Trazodone
Haloperidol
Verapamil
Loperamide

RMT*
0.604
0.641
0.670
0.795
0.808
0.897
0.910
0.965
0.982
0.991
1.042
1.062

Calibration Curve
0.198 x – 0.035
1.080 x + 0.156
0.661 x + 0.097
0.219 x + 0.828
0.493 x + 0.088
0.940 x + 0.128
2.128 x + 0.431
0.235 x + 0.017
0.379 x + 0.048
0.581 x + 0.104
0.673 x + 0.056
0.591 x + 0.044

R
0.966
0.996
0.997
0.998
0.996
0.995
0.997
0.997
0.997
0.997
0.997
0.997

*RMT – Relative Migration Time

Table 4: Linear calibration data for drugs and metabolites.

11

R2
0.934
0.993
0.995
0.996
0.993
0.991
0.993
0.995
0.994
0.994
0.994
0.993

3. H. Copley, “Validation of Capillary Electrophoresis in
Forensic Toxicological Analyses: Screening Whole
Blood for Common Basic Drugs”, A Thesis Submitted
to the College of Graduate Studies and Research inPartial Fulfilment of the Requirements for the Degreeof Master of Science in the Toxicology Graduate
Program University of Saskatchewan, Saskatoon.
(2004).

Summary and Conclusions
This article contains the information necessary to analyze
basic drugs and metabolites in forensic casework by
CESI-MS. The use of a performance standard is detailed
along with sources of the individual components. CE
and MS methods are included and explained in detail.
Steps to successfully integrate CE with MS are outlined.
The results obtained spiked urine analysis of a mixtureof drugs and metabolites with an internal standard
demonstrates the linearity and limits of detection possible
with the use of this system. The response was linear from0.25 to 50 ng per mL of urine. The LOD and LOQ wereboth 0.25 ng/mL for 12 of the 15 drugs quantitated. The
procedures, methods and protocol outlined in the article
are suitable for quantitation of forensically significant
drugs.

4. J.C. Hudson, M.J. Malcolm and M. Golin,“Advancements in Forensic Toxicology: CZE Replaces
GC/NPD as the Screen of Choice for Basic Drugs”,
P/ACE Setter Newsletter Vol. 2 Issue 4 1-5 (1998).
5. J.C. Hudson, A.F. Chen and J.D. Chapman, “DrugConfirmation with CE/MS/MS”, P/ACE Setter
Newsletter Vol. 6 Issue 1 6-10 (2002).
6. C.L. Schultz and M. Moini, “Capillary Electrophoresis
to Mass Spectrometry Interface Using a Porous
Junction.” Anal Chem. 75(9), 2188-2191 (2003).

References
1. J.C. Hudson, M. Golin and M. Malcolm, “Capillary Zone
Electrophoresis in a Comprehensive Screen for BasicDrugs in Whole Blood”, Can. Soc. Forens. Sci. J. Vol.
28, 2 137-152 (1995).

7. S. Garza, S. Chang and M. Moini, “Simplifying
CE-MS Operation. 2. Interfacing Low-Flow Separation
Techniques to Mass Spectrometry Using a Porous
Tip.” Anal Chem. 2007 79(11), 4241-4246 (2007).

2. J.C. Hudson, M. Golin, Malcolm and C.F. Whiting,“Capillary Zone Electrophoresis Comprehensive
Screen for Drugs of Forensic Interest in Whole Blood:
An Update”, Can. Soc. Forens. Sci. J. Vol. 31, 1 1-29
(1998).

8. M.E. Sharp, “Evaluation of a Procedure for Basicand Neutral Drugs: n-Butyl Chloride Extraction andMegabore Capillary Gas Chromatography”. Can. Soc.
Forens. Sci. J., Vol. 19(2), 83-101(1986).

For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. SCIEX is part of AB Sciex. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
* In development. CESI and OptiMS are for Laboratory Use Only; not for use in diagnostic procedures.
Beckman Coulter, the stylized logo and Allegra are trademarks of Beckman Coulter, Inc., and are registered with the USPTO.
CESI and OptiMS are trademarks of Beckman Coulter, Inc.
All trademarks are the property of their respective owners

View SCIEX products at www.sciex.com
Find your local office at www.sciex.com/offices

AB SCIEX Headquarters
500 Old Connecticut Path | Framingham, MA 01701 USA

For Beckman Coulter’s worldwide office locations and phone numbers,
visit “Contact Us” at www.beckmancoulter.com
Phone please508-383-7700
www.sciex.com/ce

B2011-12537

12© 2011 Beckmanwww.absciex.com
Coulter, Inc.

Printed in U.S.A.