On-line sample preconcentration prior to CE-ESI-MS/MS: quantitation of drugs of abuse in bioanalysis

Isabelle KOHLER1,2, Julie SCHAPPLER1,2, Martin GREINER3, Serge RUDAZ1,2

Isabelle.kohler@unige.ch

1School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d’Yvoy 20, 1211 Geneva 4, Switzerland, 2Swiss Centre for Applied Human Toxicology (SCAHT), University

of Geneva, CMU, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland, 3Agilent Technologies R&D and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany

The hyphenation of capillary electrophoresis (CE) with mass spectrometry (MS), particularly tandem mass spectrometry (MS/MS), is well adapted in bioanalysis for the

quantitation of drugs of abuse due to numerous advantages, such as high separation efficiency, high sensitivity, high selectivity, short analytical time and low solvent and sample

consumption. Due to the complexity of biological matrices, a sample preparation is often mandatory to lower matrix compounds which can interefere with the ionization process.

Electrospray ionization

Agilent Jet Stream Coaxial sheath liquid interface

Sheath liquid composition H2O/i-PrOH/HCOOH

50:50:0.5 (v/v/v)

Sheath liquid flow rate 5 µL/min

Drying gas temperature 200 °C

Drying gas flow rate 16 L/min

Nebulizing gas pressure 8 psi

Sheath gas temperature 200 °C

Capillary electrophoresis Electrospray ionization - tandem mass spectrometry

Sample preparation

CE Separation

MS/MS Detection

Most time consuming step

(ca. 2/3 of the analytical process)

Primary source of analytical errors

Relatively high amount of solvents required

Urine is a matrix of choice for drugs of abuse determination due to its ease of collection and relatively long detection time window. In contrast to many biological

matrices, no protein is usually present in urine, which allows for a direct injection without tedious and time-consuming off-line sample preparation (i.e., protein

precipitation, liquid-liquid extraction, solid-phase extraction, etc.).

Nevertheless, urine samples have to be diluted (5- to 10-fold) with buffer prior to CE injection to

• Lower the concentration of urinary salts

• Normalize urine pH (physiological values: 3.5-8).

The loss of sensitivity can be balanced with the implementation of an on-line preconcentration technique (sample stacking).

Drugs of

abuse

(Cocaine,

amphetamines,

opiods, etc.)

pKabasic : 6.5 – 10.5

For weak cationic compounds, mostly encountered with drugs of abuse, a pH-mediated stacking procedure is particularly well adapted.

Prior to the injection, urine samples were diluted 10-fold with background electrolyte (BGE) and water (1:1:8, v/v/v). A pH-mediated stacking

procedure was implemented to increase the loading capacity. A small preplug (0.7 % of the capillary length, Ltot) of NH4OH 7% (m/v) was injected

prior to a hydrodynamic injection of acidified urine sample, corresponding to 20.5% of Ltot.

The BGE was composed of 1 M formic acid at pH 1.8 to ensure the maximal ionization of targeted compounds for proper electrophoretic mobilities.

When applying the separation voltage, analytes under cationic

form in diluted urine migrate until they reach the strong

alkaline zone, become neutral and stack in a narrow zone at the

boundary of the sample and the alkaline plug. The latter is then

acidified by the BGE, and the analytes return at a cationic state

and begin their electrophoretic migration.

HCOOH 1M

pH 1.8

NH4OH

7%

BH+

BH+

BH+

BH+

B

B

B

B

0.7% Ltot 20.5% Ltot

Urine:BGE:H2O

1:1:8 (v/v/v)

BH+

BH+

BH+

BH+

BH+

BH+

BH+

BH+

BH+

BH+

BH+

N

N

+ + ++ - - - -

[m/z]

Calibration

within the matrix

Improvement in loading capacity is

shown for cocaine (COC) spiked at 100

ng/mL in urine:

A. Injection of 1.0% of the capillary length

100

Re

lative

in

ten

sity

[%]

m/z 304 → 182 (COC)

A. B.

1.0%

20.5% Sheath

liquid

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---

+

Triple tube

Nebulizing

gas

Tandem mass spectrometry

Agilent 6490 Triple Quadrupole

The authors wish to warmly thank Agilent Technologies for the loan of G7100 CE and 6490 Triple Quadrupole LC/MS system. Dr Marc Fathi (Laboratory of Clinical Chemistry, Geneva Hospitals, Switzerland) is acknowledged for the gift of toxicological samples. The International Separation Science Society CASSS, as well as the Centre de

Compétences en Chimie et Toxicologie Analytique (cCCTA) are gratefully thanked for financial support.

B. Injection of 20.5% of the capillary length

after a preplug of NH4OH 7% (m/v)

Conventional

hydrodynamic injection

pH-mediated

stacking procedure

CE was hyphenated to a 6490 Triple Quadrupole LC/MS system from Agilent Technologies (Palo Alto, CA, USA) via a coaxial sheath flow Agilent Jet Stream (AJS) electrospray interface (ESI).

This set-up allows for an enhanced sensitivity thanks to an improved ions’ generation and transmission with AJS and Ion Funnel technologies.

In the AJS thermal gradient focusing Technology configuration, a superheated nitrogen sheath

gas surrounds the nebulizer spray, increasing desolvation efficiency. More ions and fewer

solvent droplets enter the sampling capillary, resulting in greater signal and less noise.

The mass analyzer encompasses Ion Funnel technology, which increases the ions transmission

efficiency through the sequential pumped vacuum chambers. The ion funnel consists in a serie

of closely spaced ring electrodes whose inner diameters gradually decrease, radially confining

ions as they pass through. With this set-up, more ions can be captured from the ESI source in

comparison with the conventional skimmer, in which only a small fraction of the ion cloud

formed in the source is sampled.

AJS and MS parameters were determined by infusing (50 mbar) a set of drugs of abuse

(cocaine, codeine, methamphetamine, MDMA, and methadone) diluted at 50 µg/mL with

BGE.

The most sensitive SRM transitions were selected.

Sheath gas flow rate 3.5 L/min

Nozzle voltage 2000 V

Capillary voltage 2000 V

Fragmentor voltage 380 V

Dwell time 80 ms

Resolution 0.7 u

EMV 300 V

The quantitative performance of the CE-ESI-MS/MS method was evaluated with cocaine (COC) and methadone (MTD) selected as model compounds, and their respective deuterated analogues (d3-COC and d3-MTD) used as internal standards. The developed CE-ESI-MS/MS

method was fully validated according to SFSTP protocols and Guidance of Food an Drug Administration for bioanalytical method validation with evaluation of selectivity, response function, lower limit of quantitation (LLOQ), trueness, precision, accuracy and linearity.

Selectivity

Neat

standard

(25 ng/mL)

water

urine

spiking solution

Post-dilution

spiked matrix

(25 ng/mL)

Matuszewski et al., Anal. Chem. 75 (2003) 3019

Dilution

Matrix effect (ME)

Matrix effect (ME) was quantified to determine the influence of

potential co-migrating interferents on the ionization according to the

methodology proposed by Matuszewski et al.

ME (n = 3)

COC 87 ± 14%

MTD 81 ± 15%

9 10 11 12

VS

VS

CAL 0

CAL 0

CAL 00

CAL 00

Migration times [min]

Intensity [cps]

m/z 307 → 185 (d3-COC)

8

m/z 304 → 182 (COC)

m/z 307 → 185 (d3-COC)

m/z 304 → 182 (COC)

m/z 307 → 185 (d3-COC)

m/z 304 → 182 (COC)

0 1 2 3 4 5 9 10 11 12

Migration times [min]

VS

VS

CAL 0

CAL 0

CAL 00

CAL 00

Intensity [cps]m/z 313 → 268 (d3-MTD)

8

m/z 310 → 265 (MTD)

m/z 313 → 268 (d3-MTD)

m/z 310 → 265 (MTD)

m/z 313 → 268 (d3-MTD)

m/z 310 → 265 (MTD)

CAL 00

CAL 0

VS

d3-COC/ d3-MTD

d3-COC/ d3-MTD

COC/MTD (25 ng/mL)

Selectivity

Validation

Three independant

series

(j = 3)

Ordinary least square after square root transformation of

concentration (x) and response (y)

Relative bias

[%]

10 ng/mL 12.0 38.5

25 ng/mL 1.0 1.7

500 ng/mL -3.1 -1.8

1000 ng/mL 0.1 0.7

Repeatability/

intermediate precision

[RSD, in %]

10 ng/mL 5.7 / 5.7 21.0 / 27.8

25 ng/mL 5.0 / 5.0 7.1 / 7.1

500 ng/mL 4.1 / 4.1 2.8 / 3.6

1000 ng/mL 3.0 / 3.3 3.0 / 3.0

Lower/upper confidence limits

of the total errors

[%]

10 ng/mL -1.1 / 25.1 -25.5 / 102.5

25 ng/mL -10.5 / 12.4 -14.7 / 18.0

500 ng/mL -12.5 / 6.3 -10.1 / 6.5

1000 ng/mL -7.4 / 7.6 -6.2 / 7.6

LLOQ [ng/mL] 10 21

Range 10-1000 ng/mL 21-1000 ng/mL

Slope 0.9947 1.0074

Intercept -2.2629 -4.1128

R2 0.9977 0.9977

Trueness

Precision

Accuracy

LLOQ

Linearity

Response function

Calibration standards (CS)

(k = 3, n = 2)

Within the matrix

Validation standards (VS)

(k = 4, n = 4)

Within the matrix

60%

80%

100%

120%

140%

0 200 400 600 800 1000

Tru

en

es

s [

%]

Concentration [ng/mL]

60%

80%

100%

120%

140%

0 200 400 600 800 1000

Tru

en

es

s [

%]

Concentration [ng/mL]

Evaluation of the quantitative performance of the

developed CE-ESI-MS/MS with accuracy profiles

(α = 5%, df = k · j – n)

Inclusion of lower/upper confidence limits of the mean bias

within the acceptance limits (± 30%, Guidance of FDA) for

each level of concentration.

CE-ESI-MS/MS method accurate for the range 10-1000 ng/mL.

Exclusion of the lowest concentration (10 ng/mL) due to

unacceptable trueness and precision.

LLOQ defined at 21 ng/mL with graphical intrapolation of

absolute accurate profile.

CE-ESI-MS/MS method accurate for the range 21-1000 ng/mL.

COC and MTD contained in two toxicological samples coming from

the Laboratory of Clinical Chemistry (Geneva Hospitals, Switzerland)

were quantified with the developed method.

Because most of the variability came from repeatability, two analyses

(N = 2) were performed on each sample. Results were expressed by :

1 2 3 4 5 6 9 10 11 12

Intensity [cps]

Migration times [min]

m/z 307 → 185 (d3-COC)

m/z 304 → 182 (COC)

8

9 10 11 12

Migration times [min]

m/z 313 → 268 (d3-MTD)

m/z 310 → 265 (MTD)

8

Intensity [cps]

41.0 ± 6.4

ng/mL

462.9 ± 33.5

ng/mL

A fast, selective and sensitive CE-ESI-MS/MS method was developed for the quantitation of drugs of abuse in urine samples. Urines were simply diluted prior to the injection, avoiding a tedious offline sample preparation. A pH-mediated stacking procedure was implemented to increase the loading capacity (20.5% of the capillary length) during

injection. The developed CE-ESI-MS/MS was validated with two model compounds. COC was fully validated over the range of concentrations of 10-1000 ng/mL with accuracy included within the ± 30% acceptance limits, as for MTD in the concentrations range of 21-1000 ng/mL. Finally, this method was applied to real urine samples from drugs

consumers. Due to its sensitivity and speed, the developed CE-ESI-MS/MS method has proven to be fully applicable for high throughput in routine analysis.

22

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Mean result

Number of analyses

Inter-series variance

Repeatability

Student constant dependant on df and α