Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 1749–1757

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1983

Quantitative determination of cetirizine enantiomers

in guinea pig plasma, brain tissue and microdialysis

samples using liquid chromatography/tandem

mass spectrometry

Anubha Gupta1*, Britt Jansson1, Pierre Chatelain2, Roy Massingham2

and Margareta Hammarlund-Udenaes1

1Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Uppsala University, Box 591,

SE-751 24 Uppsala, Sweden2UCB S.A. Pharma Sector, Braine-l’Alleud, Belgium

Received 6 April 2005; Revised 26 April 2005; Accepted 26 April 2005

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric

detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-

cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective

separation was achieved on an a1-acid glycoprotein column within 14 min for all methods. A cetir-

izine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were

detected by multiple reaction monitoring using transitions m/z 389.1! 200.9 and 396.1! 276.1,

respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea

pig plasma, the assay was linear over the range 0.25–5000 ng/mL for both S-CZE and R-CZE, with a

lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples,

the assays were linear over the range 2.5–250 ng/g and 0.25–50 ng/mL, respectively, and the LLOQ

values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were

�7.1% and �12.6%, respectively, and the intra- and inter-day accuracy varied by less than �8.0%

and �6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.

Copyright # 2005 John Wiley & Sons, Ltd.

Cetirizine (CZE) is a non-sedative second-generation H1-

antihistamine widely prescribed for the treatment of allergic

disorders. It exists as a racemic mixture of levocetirizine

(R-CZE) and dextrocetirizine (S-CZE) (Fig. 1). The antihista-

minic properties of CZE are attributable to R-CZE.1 The

main side effects of the first-generation antihistamines are

sedation, drowsiness and impaired performance, which

are caused by their action on histamine H1-receptors in the

brain. The second-generation antihistamines have lower inci-

dence of these side effects than the first-generation drugs

because of lower brain penetration. It has been suggested

that the reduced brain penetration of CZE could be the result

of P-glycoprotein-mediated efflux at the blood-brain barrier

(BBB).2,3 Because of the probable involvement of an active

efflux process, the transport of CZE across the BBB could be

stereoselective and therefore is worthy of investigation.

Microdialysis is a valuable tool for studying the transport

of drugs across the BBB.4 It offers the advantage of the ability

to estimate unbound drug concentrations in both blood and

brain in one animal over time. Measuring the concentration of

unbound drug in the blood and the brain interstitial fluid

(ISF), and the total concentration of drug in the brain, allows

characterization of both transport into and distribution

within the brain. The guinea pig is preferable to the rat as a

model species because of the higher brain concentrations of

H1-receptors, and is often used in work on antihistamines.5

Investigation of the brain transport of CZE enantiomers

using microdialysis techniques requires an analytical method

capable of detecting the CZE enantiomers in plasma, whole

brain tissue, and in Ringer’s solution (perfusion fluid). CZE is

highly protein-bound, which will result in lower unbound

concentrations in the blood. It has been reported that only a

small proportion of the available CZE enters the brain,

resulting in even lower unbound concentrations of the two

enantiomers in brain ISF.6 Therefore, a highly sensitive

analytical method was a prerequisite for this study, and mass

spectrometric detection was an obvious choice. Bakhtiar et al.

have reviewed the use of mass spectrometry with enantio-

selective liquid chromatography.7 Other challenges with

such an analytical method involve the handling of the small

sample volumes (8–15 mL) obtained from the microdialysis

experiment. The Ringer’s solution used in the microdialysis

experiments contains high salt levels, which causes signal

depression in electrospray ionization (ESI) mass spectro-

metry. In addition, only a limited volume of blood can be

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: A. Gupta, Division of Pharmacokinetics andDrug Therapy, Department of Pharmaceutical Biosciences,Uppsala University, Box 591, SE-751 24 Uppsala, Sweden.E-mail: [email protected]

drawn from each guinea pig without affecting the home-

ostasis of the animal.

Several methods for the quantification of racemic CZE

have been described in the literature.8–14 However, to date,

only Choi et al. have described a stereoselective method,

using UV detection, for the determination of CZE in rat

plasma15 and human urine.16 A chiral a1-acid glycoprotein

(AGP) column was used by these workers to separate the

enantiomers. A volume of 100 mL was used for rat plasma,

and the lower limit of quantification (LLOQ) was 800 ng/mL.

For human urine samples, a 5 mL aliquot was used, and the

LLOQ was 400 ng/mL. The internal standard was roxatidine,

eluting at 32 min. This method, however, has poor sensitivity

and takes a relatively long time.

Four non-enantioselective methods which use mass spec-

trometric detection for the estimation of cetirizine concentra-

tions have also been described.8–10,13 Eriksen et al.9 used a

sample volume of 250 mL and validated the method with an

LLOQ of 5 ng/mL. Li et al.10 and Song et al.13 achieved an

LLOQ of 1 ng/mL, using a sample volume of 100mL. De Jager

et al.8 reported an LLOQ of 0.5 ng/mL, using a 100 mL plasma

sample.

In this paper we describe the development and validation

of three enantioselective liquid chromatographic assay

methods using tandem mass spectrometric detection (LC/

MS/MS) for the determination of S- and R-CZE in guinea pig

plasma (plasma method), guinea pig brain tissue (brain

method), and microdialysis samples from guinea pig blood

and brain ISF (Ringer method). Enantioselective separation

was achieved within 14 min for all three methods. Because of

the sensitivity and specificity of MS, it was possible to both

keep the sample volume small and retain a high level of

sensitivity. Preparation of the samples using protein pre-

cipitation was simple and rapid. The methods were used to

analyze guinea pig plasma and brain tissue samples and

samples from microdialysis of blood and brain ISF to in-

vestigate the possible stereoselective transport of CZE across

the BBB.

EXPERIMENTAL

Chemicals and reagentsRacemic CZE, S-CZE, and the internal standard (IS, ucb

20028), were provided by UCB Pharma (Braine-l’Alleud,

Belgium). Acetonitrile (ACN) and 2-propanol (LiChrosolve,

gradient grade), ammonia and ammonium acetate (analytical

grade) were obtained from Merck (Darmstadt, Germany).

Methanol (HPLC gradient grade) was purchased from

J.T. Baker (Deventer, The Netherlands). MQ water, which

is in-house deionized water, further purified in a Milli-Q

Academic system (Millipore, Bedford, MA, USA), was used.

Ringer’s solution (KCl 33 mg, CaCl2 H2O 330 mg, NaCl

8.6 g, sterilized water for injection Ph. Eur. ad 1000 mL,

pH ca. 6, osmolarity 290 mosm/kg H2O; Fresenius Kabi

Norge AS) was obtained from the Uppsala Hospital Phar-

macy, Uppsala, Sweden. Bovine serum albumin (BSA, initial

fractionation by heat shock) was provided by Sigma Chemi-

cal Co. (St. Louis, MO, USA). Blank guinea pig plasma and

brain tissue were obtained from male Dunkin Hartley guinea

pigs (Charles River, France).

InstrumentationChemical analyses were performed using an LC/MS/

MS system consisting of two Shimadzu LC-10AD pumps

(Shimadzu, Kyoto, Japan), a Triathlon 900 autosampler

(Spark Holland, The Netherlands) equipped with a 100 mL

loop and an extra six-port valve, a pre-filter (0.5mm;

Upchurch Scientific Inc, Hagersten, Sweden), an AGP col-

umn (150� 4.0 mm; ChromTech, Hagersten, Sweden), and a

Quattro Ultima triple-quadrupole mass spectrometer (Micro-

mass, Manchester, UK).

Liquid chromatographyFor the plasma and brain methods, the mobile phase con-

sisted of 6.5% ACN in 10 mM ammonium acetate, pH 7.0

(adjusted with 1 % ammonia). The column was maintained

at 308C in a water bath (type JB1, Grant Instruments,

Cambridge, UK).

For the Ringer method, the column was maintained at

room temperature and 5.5% ACN was used in the mobile

phase. A switch was used after the analytical column to avoid

salt overload in the mass spectrometer. Two pumps were

used; one was connected to the column through the auto-

sampler and the other was connected directly to the extra

six-port valve. During the time period 0–6.5 min after

sample injection, the flow of the mobile phase from the

column was discarded and the flow of mobile phase from

the other pump entered the mass spectrometer. During the

period 6.5–14 min, the flow from the column entered the

mass spectrometer.

The mobile phase was pumped at a flow rate of 0.9 mL/

min, generating a pressure of 100 bar. The flow was split

to 250–270 mL/min before entering the mass spectrometer.

The order of elution of the enantiomers was determined by

injection of pure S-CZE.

Mass spectrometeryThe triple-quadropole mass spectrometer was used in

the positive ESI mode. The measurements were made at

NO

O

O

O

ucb 20028

ONN

Cl

O

OH*

Cetirizine

Figure 1. Chemical structures of cetirizine and the internal

standard (ucb 20028). The asterisk represents the chiral

center.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 1749–1757

1750 A. Gupta et al.

4008C desolvation temperature, 1308C source temperature,

200 L/h cone gas, 1000 L/h desolvation gas and a collision

gas pressure of 3� 10�3 Torr. The capillary voltage was

1.5 kV and the cone voltage was 40 V. The collision energy

was set to 20 eV for CZE and 25 eV for the IS. The resolution

was set at 0.9 u at half height for Q1 and Q3. The dwell time

for all channels was set to 0.5 s and inter-channel delay was

0.1 s. To monitor the multiple reactions (MRM), the MS/MS

parameters were optimized to provide the highest sensitivity

by directly infusing (200 mL/min) working solutions con-

taining CZE and IS (100 ng/mL in the mobile phase) using a

Harvard 22 syringe pump (Harvard Apparatus Inc., Hollis-

ton, USA). The transitions that gave the most intense signals

were m/z 389.1! 200.9 for CZE and 396.1! 276.1 for the IS

(Fig. 2).

Preparation of standard and qualitycontrol (QC) samplesStock solutions of CZE and IS (1 mg/mL) were prepared in

MQ water and stored at 48C, protected from light.

For the plasma method, blank guinea pig plasma was

spiked with the stock CZE solution. The standard curve

consisted of ten samples containing each enantiomer in

the range of 0.25–5000 ng/mL. Three QC samples, low-

concentration (QCL, 0.75 ng/mL), medium-concentration

(QCM, 250 ng/mL), and high-concentration (QCH, 3710 ng/

mL), were prepared.

For the brain method, the blank brain sample was pre-

pared by homogenizing blank brain tissue with a 4-fold

volume (w/v) of saline. The standard samples were prepared

by spiking blank brain tissue samples with the stock

CZE solutions before homogenization; the brain was then

homogenized with a volume of saline equivalent to a 4-fold

volume (w/v) minus the volume of the added analyte

solution, and the homogenate was used for further proces-

sing. The standard curve for guinea pig brain composed of

seven samples containing each enantiomer in the range of

2.5–250 ng/g brain. Three QC samples (concentrations 12.5,

75 and 188 ng/g brain) were prepared.

For the Ringer method, BSA was soaked in Ringer’s

solution overnight and the volume was adjusted the next

day to give a blank of 0.5% BSA in Ringer’s solution. This

solution was then spiked with the stock CZE solutions to give

eight standard samples in the range of 0.25–50 ng/mL for

each enantiomer. Three QC samples (concentrations 0.38,

7.5 and 38 ng/mL) were prepared.

The standards and QC samples of guinea pig plasma,

guinea pig brain homogenate, and Ringer-BSA solution were

stored at �208C until analysis. The working solution for

the IS was prepared daily in ACN.

Study sample collectionThe guinea pigs were anaesthetized by inhalation of

Enflurane1 (2.5% balanced with 1.5 L/min oxygen and

1.5 L/min nitrous oxide) and 0.25 mL of Dormicum (midazo-

lam 5 mg/mL) intraperitoneally. FEP tubings were inserted

into the left jugular vein for drug administration and into

the left common carotid artery for blood sampling. The blood

probe (CMA/20, polycarbonate, 20 kDa cutoff, 10 mm) was

inserted into the right jugular vein. A CMA/12 guide cannula

with a dummy probe was implanted in the brain stereotaxi-

cally and fixed to the skull with a screw and dental cement.

When the cement had stiffened, the dummy probe was

replaced with the 3 mm (CMA/12) brain probe. The guinea

pig was placed in a CMA/120 system for freely moving

animals with free access to water and food, and the experi-

ment was performed approximately 24 h later. On the day

of the experiment, each guinea pig received a 60 min intrave-

nous (i.v.) infusion of cetirizine (2.7 mg/kg). The microdialy-

sis blood and brain ISF samples were obtained by collecting

dialysate from the microdialysis probes in fractions of 15 min.

The probes were continuously perfused with Ringer’s solu-

tion containing BSA from the beginning of the infusion

up to 360 min. The blood samples were collected at pre-

defined time points, and the plasma was separated by centri-

fugation for 5 min at 10 000 rpm. The brain tissue samples

were obtained by decapitating the guinea pig at the end

of the experiment. All the samples were stored at �208Cuntil analysis.

Sample preparation

Plasma methodPlasma (50mL) was precipitated with ACN (100mL) con-

taining IS at a concentration of 100 ng/mL in a 1.5 mL poly-

propylene Eppendorf tube (Brand, Wertheim, Germany).

The sample was vortexed for 5 s and centrifuged at

100 150 200 250 300 350 400m/z0

100

%

276

176167 202

396

338

50 100 150 200 250 300 350 400m/z0

100

%

201

201

50166

201

389

MRM 389.1 200.9

MRM 396.1 276.1

(a)

(b)

Figure 2. Product ion MS/MS spectra of [MþH]þ ions of (a)

IS and (b) CZE.

Cetirizine enantiomers in guinea pig plasma and brain ISF and homogenate 1751


10 000 rpm for 5 min using a Force 7 centrifuge (Denver

Instrument Co., Denver, CO, USA). Then, 50 mL of the super-

natant were evaporated at 408C under N2 gas, and the residue

was dissolved in 500mL mobile phase by vortex mixing

and sonication. Finally, 60 mL of the reconstituted sample

were transferred to a polypropylene autosampler vial and a

volume of 50 mL was injected, using the partial loop fill tech-

nique, onto the LC/MS/MS system.

Brain methodA volume of 50mL of the brain homogenate was sampled and

processed as in the plasma method but with the following

modifications: the concentration of IS used was 5 ng/mL,

the residue was dissolved in 1000 mL mobile phase, and a

volume of 40 mL was injected.

Ringer methodSamples from the microdialysis experiment were collected

directly into polypropylene autosampler vials. The vials

were weighed before and after sample collection. The volume

collected varied from 6 to 8mL. A volume of 8mL of each

standard or QC sample was transferred into a polypropylene

autosampler vial. The microdialysis study, standard and QC

samples were precipitated with 100 mL ACN containing IS

(2 ng/mL), as described above. The supernatant (100mL)

was transferred to an Eppendorf tube and evaporated. The

Figure 3. MRM chromatograms of (a) a blank guinea pig plasma sample, (b) a spiked

guinea pig plasma sample containing 125 ng/mL of each of S- and R-CZE plus 100 ng/

mL of the IS, and (c) a plasma sample from a guinea pig dosed with CZE where the

measured concentrations of S- and R-CZE were 190 and 583ng/mL, respectively.



residue was reconstituted in 50 mL mobile phase and a

volume of 40 mL was injected.

For all three methods, the autosampler was thermostated

at 88C. A wash solution (50% ACN) of 1500mL was used

between the injections.

ValidationLinearity, precision, accuracy, specificity and lower limit

of quantification (LLOQ) were assessed. The linearity

was determined from peak area ratios (analyte/IS) as a

function of analyte concentration, using linear regression.

For the plasma method, the calibration curve was

forced through the origin. For both the brain and Ringer

methods a weighting factor of 1/y2 was used and the

calibration curve was not forced through the origin.

Peak areas were measured using Masslynx software,

version 4.0, individually checked and rectified manually if

required.

The precision was assessed as the coefficient of variation

(CV) of the back-calculated concentrations of the three

QC samples. The accuracy was calculated as the percent

deviation of the analyzed concentration from the nominal

value of the three QCs. For intra-day precision and accuracy,

one calibration curve and six independent sets of three QCs

were analyzed on one occasion. The inter-day precision

and accuracy were determined by two methods, either by

analyzing six independent sets of three QCs on two

additional occasions, or by pooling the two sets of three

QCs during six runs. During these runs, QC samples were

interspersed with the unknown study samples. The LLOQ

was set at the concentration that could be analyzed with a

precision of �20% and an accuracy of �20%.

(a)

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00Time0

100

%

0

100

%

CZE_111_74 MRM of 2 Channels ES+396.15 > 276.1

597


1.20e3

(b)

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00Time0

100

%

0

100

%


9.52e3


1.58e4

(c)

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00Time0

100

%

0

100

%


8.94e3


1.61e4

S-CZE

S-CZE R-CZE

R-CZE

IS

IS

Figure 4. MRM chromatograms of (a) blank guinea pig brain tissue sample, (b) a spiked

standard sample containing 50 ng/g of each of S- and R-CZE plus 5 ng/mL of the IS, and (c)

a homogenized brain tissue sample from a guinea pig dosed with CZE where the measured

concentrations of S- and R-CZE were 91 and 56 ng/g, respectively.



Application of the analytical methodThe validated methods were applied to the analysis of

guinea pig plasma, brain and microdialysis blood and brain

ISF samples obtained from the experimental study of the

transport of CZE enantiomers across the BBB.

RESULTS AND DISCUSSION

Chromatographic conditionsTo optimize the chromatographic separation of CZE enantio-

mers, a mobile phase giving a resolution of 1.8 or higher and a

retention time less than 15 min was selected. The enantiose-

lective separation on the AGP column was affected by the

type (ACN, methanol or 2-propanol) and concentration of

organic modifier, the pH of the mobile phase, and the buffer

concentration. Higher concentrations of organic modifier

resulted in shorter retention times. Increases in the pH

improved the resolution of the peaks. The recommended

pH range for the AGP column is 4 to 7.17 Adequate separa-

tion of the enantiomers within a reasonable time was

achieved with a mobile phase of pH 7.0 containing 6.5%

ACN. A reduction in retention time after injecting ap-

proximately 3500 samples onto the AGP column indicated

that the number of theoretical plates was reduced. The

ACN content was therefore lowered to achieve the same

retention times.

Figure 5. MRM chromatograms of (a) blank Ringer’s solution with BSA, (b) a spiked

Ringer-BSA solution sample containing 25 ng/mL of each of S- and R-CZE plus 2 ng/mL of

the IS, and (c) a Ringer-BSA solution sample from the brain tissue of a guinea pig dosed

with CZE where the measured concentrations of S- and R-CZE were 0.80 and 0.55 ng/mL,

respectively.



In the previously reported enantioselective assay for CZE,

the mobile phase used was 5% ACN in 10 mM phosphate

buffer, at pH 7.15,16 In our study, ammonium acetate was

selected as the buffer because of its compatibility with LC/

MS systems.7 Buffer concentrations of 5–20 mM were

investigated. At pH 7, a buffer concentration of 10 mM

gave better resolution than 5 mM. Ammonium acetate

concentrations of 20 mM did not significantly influence the

resolution of the peaks, but the response of the mass

spectrometer was half that seen using 10 mM. On the basis

of these observations, the mobile phase composition was set

to 6.5% ACN in 10 mM ammonium acetate, at pH 7.0. A

column temperature of 308C resulted in decreased retention

times without compromising the peak resolution but, for the

Ringer method, the high salt concentration affected the signal

of S-CZE. By maintaining the column at room temperature

and using 5.5% ACN in the mobile phase for the Ringer

method, the retention times for the CZE enantiomers were

increased and the salts were eluted before the peaks.

Representative chromatograms for the plasma method, the

brain method and the Ringer method are shown in Figs. 3–5.

S-CZE, R-CZE and IS were eluted with retention times of

approximately 6.9, 8.7, and 7.9 min, respectively, for the

plasma samples; at 6.5, 8.2, and 7.7 min, respectively, for the

brain samples; and at 8.6, 11.4, and 11.1 min, respectively,

for the microdialysis samples.

Sample preparationVarious methods of sample preparation for the estimation

of CZE have been reported in the literature. These

include liquid-liquid extraction,11,14 solid-phase extraction

(SPE),9,10,13 and protein precipitation.8,12,15 The methods

screened in this study included SPE and protein precipita-

tion with acetonitrile. For a sensitive protein column an

SPE method would have been preferable, but CZE was

found to be adsorbed to the SPE assembly (C18 Sep-Pak1).

Protein precipitation with acetonitrile was a more rapid

and simple preparation approach, but this method can result

in high peak suppression. In the plasma method the sample

was diluted 30 times. The response of CZE and IS, obtained

from the extracted spiked plasma sample, was similar to

that from spiked mobile phase, indicating that suppression

effects were not significant. However, a depression in the

baseline was observed at 4.5–5 min in the chromatogram of

a blank plasma sample, shown in Fig. 3.

The method used for the brain homogenate samples was

that reported by Chen et al.2 The effect of the matrix was more

pronounced for the brain samples than the plasma samples.

When the residue was reconstituted in 500mL mobile phase,

the signal for S-CZE was depressed and the peak area for

S-CZE was approximately half that for R-CZE. When the

residue was dissolved in 1000mL of mobile phase, this

difference disappeared.

The samples obtained in microdialysis studies are usually

protein-free, allowing direct injection into the chromato-

graphic system. However, for CZE, 0.5% BSA was added to

the Ringer’s solution to prevent the adsorption of CZE onto

the materials used in the microdialysis experiments. For this

reason, the microdialysis samples needed to be prepared

before they were injected into the column.

ValidationThe standard curve obtained using the plasma method was lin-

ear over the range of 0.25–5000 ng/mL for both S- and R-CZE

(Fig. 6(a)). The three standard samples with the highest con-

centrations (5000, 4500 and 2500 ng/mL) and the QCH were

dissolved in 1000mL of mobile phase to avoid overloading

the mass spectrometer. All the unknown study samples that

showed a peak area larger than that of the standard sample

with a concentration of 2500 ng/mL were diluted twice before

re-injection. The standard curve obtained using the brain

method was linear over the range of 2.5–250 ng/mL and that

using the Ringer method was linear over 0.25–50 ng/mL. Both

these calibration curves were made with a weighting factor

of 1/y2, which resulted in an even residual distribution,

giving similar emphasis to all concentrations over the entire

standard curve range. The coefficient of determination was

greater than 0.996 for both enantiomers in all methods.

(a)

S-CZEy = 0.0051xR2 = 0.9996

R-CZEy = 0.0055xR2 = 0.9998

0

5

10

15

20

25

30

0 1000 2000 3000 4000 5000 6000

Concentrat ion (ng/mL)

Rat

io

(b)

S-CZEy = 0.0358x + -0.0049

Coefficient of Determinat ion = 0.9984

R-CZEy = 0.0363x + -0.0037

Coefficient of Determination = 0.9983

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0 50 100 150 200 250 300

Concentrat ion (ng/mL)

Rat

io

(c)

S-CZEy = 0.0269x + - 0.0002


R-CZEy = 0.0305x + - 0.0012


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 50 60

Concentration (ng/mL)

Rat

io

Figure 6. Standard curves for (a) the plasma method, (b)

the brain method, and (c) the Ringer method. S-CZE and R-

CZE are represented by diamonds and squares, respectively.

The response is expressed as the ratio of the area of the

analyte to that of the internal standard.



The precision and accuracy of the plasma, brain and Ringer

methods are summarized in Tables 1–3. For the plasma

method, the intra-day precision was �7.1% and the accuracy

deviated by less than �3.1% of the nominal value. In this

method, the inter-day precision was�12.6% and the accuracy

deviated by less than �1.8% of the nominal value. For the

brain method, the intra- and the inter-day precisions were

�4.2% and �6.8%, respectively. The intra- and inter-day

accuracies deviated by less than �8.0% and �6% of the

nominal value, respectively. For the Ringer method, the intra-

day precision was�6.5% and the accuracy varied by less than

�5.2% of the nominal value. The inter-day precision was

�10% and the accuracy varied by less than �5.9% of the

nominal value.

The enantioselective assay of CZE was specific, as blank

plasma, brain and Ringer’s solution with 0.5% BSA samples

were free from any direct interferences in the MRM channels

at the retention times for IS, S-CZE and R-CZE (Figs. 3–5).

Table 2. Intra- and inter-day precision and accuracy of cetirizine enantiomers in guinea pig brain tissue. The enantiomers were

separated using an a1-acid glycoprotein (AGP) column prior to tandem mass spectrometry. Ucb 20028 was used as internal

standard

QCH (188 ng/mL) QCM (75.8 ng/mL) QCL (12.6 ng/mL) LLOQ (2.51 ng/mL)

S R S R S R S R

Intra-dayObserved conc.(ng/mL) 192 191 81.4 81.9 12.9 12.9 2.54 2.61CV (%) 2.9 2.9 2.6 2.4 3.4 4.2 10.2 7.6Accuracy (%) 1.8 1.7 7.3 8.0 1.8 1.8 1.3 4.0n 6 6 6 6 6 6 6 6Inter-dayObserved conc.(ng/mL) 190 190 80.2 80.4 13.0 13.1CV (%) 2.6 2.9 6.4 6.8 4.9 4.8Accuracy (%) 1.0 1.0 5.8 6.0 3.0 3.5n 18 18 18 18 18 18

Table 1. Intra- and inter-day precision and accuracy of cetirizine enantiomers in guinea pig plasma. The enantiomers were

separated using an a1-acid glycoprotein (AGP) column prior to tandem mass spectrometry. Ucb 20028 was used as internal

standard

QCH (3710 ng/mL) QCM (251 ng/mL) QCL (0.75 ng/mL) LLOQ (0.25 ng/mL)

S R S R S R S R

Intra-dayObserved conc.(ng/mL) 3690 3780 243 251 0.73 0.76 0.29 0.28CV (%) 3.4 2.3 2.7 0.7 7.1 3.7 10.8 12.7Accuracy (%) �0.6 1.9 �3.1 0.0 �3.0 0.7 15.0 12.4n 6 6 6 6 6 6 6 6Inter-dayObserved conc.(ng/mL) 3730 3760 248 245 0.76 0.77CV (%) 3.95 3.57 6.61 6.19 11.9 12.6Accuracy (%) 0.51 1.28 �0.82 �2.31 0.88 1.82n 12 12 12 12 12 12

Table 3. Intra- and inter-day precision and accuracy of cetirizine enantiomers in Ringer’s solution with bovine serum albumin

(BSA). The enantiomers were separated using an a1-acid glycoprotein (AGP) column prior to tandem mass spectrometry.

Ucb 20028 was used as internal standard

QCH (37.8 ng/mL) QCM (7.56 ng/mL) QCL (0.38 ng/mL) LLOQ (0.25 ng/mL)

S R S R S R S R

Intra-dayObserved conc.(ng/mL) 37.3 37.6 7.50 7.57 0.39 0.36 0.26 0.25CV (%) 1.4 1.0 2.3 1.7 6.5 6.0 13.3 10.0Accuracy (%) �1.4 �0.6 �0.8 0.1 3.6 �5.2 3.8 �1.5n 6 6 6 6 6 6 6 6Inter-dayObserved conc.(ng/mL) 38.8 39.0 7.78 7.66 0.40 0.40CV (%) 3.9 3.8 2.1 2.5 10.0 9.0Accuracy (%) 2.6 1.9 2.9 1.4 5.9 5.1n 12 12 12 12 12 12



The LLOQ for the CZE enantiomers for all the methods

was set to the lowest concentration of the calibration curve

ranges, since the precision and accuracy were sufficient to

fulfill the acceptance criteria (Tables 1–3). The CV, deter-

mined from six replicate samples, was below 13%, and the

accuracy varied by less than �15% of the nominal value for

both enantiomers in all matrices.

CONCLUSIONS

Chiral LC/MS/MS (ESI) methods to determine S- and R-CZE

in guinea pig plasma, brain tissue and microdialysis samples

were developed and validated. The LLOQ was 0.25 ng/mL

for the CZE enantiomers using only 50 mL plasma samples.

The LLOQ for the microdialysis samples was also 0.25 ng/

mL using a sample volume of approximately 8 mL. Because

of a high dilution factor, the LLOQ for the brain tissue sample

was 2.5 ng/g brain. The run time was no more than 14 min for

any of the methods. These validated methods were success-

fully applied to a preclinical study investigating the BBB

transport of CZE enantiomers in the guinea pig.

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Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and...

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