Two-dimensional chromatography method applied to the enantiomeric determination of lansoprazole in human plasma by direct sample injection

Article abstract of DOI:10.1002/chir.20701

A two-dimensional HPLC method based on the direct injection of biological samples has been developed and validated for the determination of lansoprazole enantiomers in human plasma. The lansoprazole enantiomers were extracted from the biological matrix using an octyl restricted access media bovine serum albumin column (C₈ RAM BSA) and the enantioseparation was performed on an amylose tris(3,5-dimethoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV detection at 285 nm. Analysis time was 25 min with no time spent on sample preparation. The method was applied to the analysis of the plasma samples obtained from nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed.

Full text of DOI:10.1002/chir.20701

CHIRALITY 22:35–41 (2010)
Two-Dimensional Chromatography Method Applied
to the Enantiomeric Determination of Lansoprazole
in Human Plasma by Direct Sample Injection
RICARDO F. GOMES, NEILA M. CASSIANO, J. PEDRAZZOLI Jr., AND QUEZIA B. CASS1*
1
1
2
1
Departamento de Qu ı´ mica, Universidade Federal de S a˜ o Carlos, S a˜ o Carlos, S a˜ o Paulo, Brazil
Faculdade de Medicina, Unidade Integrada de Farmacologia e Gastroenterologia, Universidade S a˜ o Francisco,
Bragan c¸ a Paulista, S a˜ o Paulo, Brazil
2
ABSTRACT
A two-dimensional HPLC method based on the direct injection of bio-
logical samples has been developed and validated for the determination of lansoprazole
enantiomers in human plasma. The lansoprazole enantiomers were extracted from the
biological matrix using an octyl restricted access media bovine serum albumin column
(
C RAM BSA) and the enantioseparation was performed on an amylose tris(3,5-dime-
8
thoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV
detection at 285 nm. Analysis time was 25 min with no time spent on sample prepa-
ration. The method was applied to the analysis of the plasma samples obtained from
nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and
was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed.
Chirality 22:35–41, 2010. VV C 2009 Wiley-Liss, Inc.
KEY WORDS: enantioseparation; restricted access media (RAM); column-switching;
biological fluid; polysaccharide chiral phases
INTRODUCTION
up and a polysaccharide column, under reversed-phase
mode of elution, in the second dimension for the enantio-
separation.
The validated method was applied to the analysis of the
plasma samples obtained from nine Brazilian volunteers
who ingested 30 mg capsules of racemic lansoprazole.
The chiral compound lansoprazole (see Fig. 1), 2-[(3-
methyl-4-(2,2,2-trifluoethoxy)-2-pyridinyl)methyl]sulfinyl-
benzimidazole is one of the proton pump inhibitors (PPIs)
that inhibits gastric acid secretion through an interaction
1
1
1
with (H /K )-ATPase in gastric pariental cells. This
drug, although it has an asymmetric sulfur in its chemical
structure, is clinically administered as a racemic mixture
of (R)-(1)- and (S)-(2)-enantiomers and is frequently pre-
scribed for the treatment of acid-related conditions such as
gastric and duodenal ulcers, Zollinger–Ellison syndrome,
MATERIALS AND METHODS
General
Acetonitrile was HPLC grade and purchased from Mal-
linckrodt Baker (St. Louis, MO). Water used for the mo-
bile phase was purified using a Milli-Q system (Millipore,
Ribeir ˜a o Preto, SP, Brazil).
Bovine serum albumin was purchased from Sigma (frac-
tion V powder minimum 98%; St. Louis, MO). Glutaralde-
hyde, potassium dihydrogen phosphate, and sodium boro-
hydride were from Merck (Darmstadt, Germany).
The racemic lansoprazole standard was supplied by
Boehringer Ingelheim do Brasil (S ˜a o Paulo, SP, Brazil).
2
and other hypersecretory diseases. Following oral admin-
istration, lansoprazole is extensively metabolized in the
liver to 5-hydroxylansoprazole and lanzoprazole sulfone,
which are mainly catalyzed by CYP2C19 and CYP3A4,
3
respectively.
A significant number of analytical HPLC methods have
been described for the determination of lansoprazole enan-
4
–9
tiomers in biological fluids. All of them, however, used
off-line sample clean-up procedures before analysis such
as liquid–liquid or solid-phase extraction. The main restric-
tion of these traditional methods is the time spent on sam-
ple preparation.
Contract grant sponsors: Funda c¸ ˜a o de Amparo a Pesquisa do Estado de
S ˜a o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cient ı´ fico e
Tecnol o´ gico (CNPq), Coordena c¸ ˜a o de Aperfei c¸ oamento Pessoal de N ´ı vel
Superior (CAPES).
This work reports the first HPLC method for the analy-
sis of plasma levels of lansoprazole’s enantiomers by direct
*
Correspondence to: Quezia B. Cass, Departamento de Qu ´ı mica, Universi-
dade Federal de S ˜a o Carlos, Cx. Postal 676, S ˜a o Carlos 13565-905, SP,
sample injection. This was achieved by the use of a two- Brazil. E-mail: quezia@pq.cnpq.br
Received for publication 4 July 2008; Accepted 13 December 2008
DOI: 10.1002/chir.20701
Published online 19 February 2009 in Wiley InterScience
dimensional chromatography system using an octyl re-
stricted access media bovine serum albumin column (C8
RAM BSA) in the first dimension for extraction and clean- (www.interscience.wiley.com).
VV C 2009 Wiley-Liss, Inc.

3
6
GOMES ET AL.
prepared at the UFSCar laboratory as previously
1
2,13
described.
Sample Preparation
Stock solutions of (6)-lansoprazole were prepared by
dissolving the drug in methanol to a final concentration of
2
00 lg/ml. From these stock solutions, eight calibration
standard solutions containing 2.0, 4.0, 8.0, 12.0, 20.0, 25.0,
0.0, and 40.0 lg/ml and three quality controls (QC) solu-
3
tions at concentrations of 4.8, 24.0, and 36.0 lg/ml were
prepared in methanol. To prepare the spiked plasma sam-
ples, aliquots (20 ll) of the appropriated solution were
placed in a culture tube and the solvent was evaporated
Fig. 1. Chemical structure of lansoprazole.
1
Ogastro capsules from Abbott Laborat o´ rios do Brasil under a stream of nitrogen. The dried analytes were recon-
S ˜a o Paulo, SP, Brazil) containing 15 mg of lansoprazole stituted using 200 ll of plasma (or mobile phase as the sol-
were purchased at a local drugstore.
vent for the extraction and transfer evaluation) and the sol-
(
The enantiomers of lansoprazole were obtained, as utions were vortex-mixed for 15 sec. A 180 ll aliquot was
enriched mixture, by chiral separation using a amylose transferred to autosampler vials and 100 ll were injected
tris[(S)-1-phenylethylcarbamate)] coated (20% w/w) onto onto the HPLC system.
˚
APS-Nucleosil (7 lm particle size and 500 A pore size)
(
Column-Switching Procedure
150 mm 3 4.6 mm I.D.) as stationary phase and hex-
ane:ethanol (70:30 v/v) as mobile phase. The optical rota-
tion of the separated enantiomers was measured in etha- the RAM and the chiral columns is illustrated in Figure 2.
nol as reported elsewhere using a Perkin–Elmer Model The position of the column-switching device alternated
41 polarimeter. The absolute configurations for the PPIs between Positions 1 and 2 and was controlled through the
The column-switching system used for the coupling of
1
0
2
1
1
have been reported and were used to correlate to the timed events using the Class-VP Software. The time
optical rotation obtained of the isolated enantiomers with sequence used is listed in Table 1.
the highest enantiomeric purity. Then, it was possible to
determine the enantiomeric elution order by injection of the plasma sample was injected into the RAM column.
R)-(1) and (S)-(2)-lansoprazole on a chiral column of The six-port valve remained in this position for 9.60 min
Initially, the column-switching was set to Position 1 and
(
amylose tris(3,5-dimethoxyphenylcarbamate) coated (20% while the macromolecules were discharged into the waste
˚
w/w) onto APS-Nucleosil (7 lm particle size and 500 A and the analytes were retained on the hydrophobic phase
pore size) (150 mm 3 4.6 mm I.D.) using acetonitrile: of RAM column. At the same time, the analytical column
water (35:65 v/v) at a flow rate of 1.0 ml/min.
was conditioned with the mobile phase delivered by
1
The administration of the Ogastro capsules and collec- Pump 2. Then, the valve changed to Position 2 directing
tion of blood samples from the volunteers were made at the flow from the waste to the analytical column. The
S ˜a o Francisco University Hospital, Bragan c¸ a Paulista, SP, transfer of the effluent fraction containing the analyte took
Brazil. Nine healthy volunteers were used in the study. between 9.6 and 12.5 min.
Pooled control human plasma was also supplied by the
University Hospital and stored at 2208C until use.
At the end of this time, the valve was switched back to
Position 1 for cleaning and conditioning the RAM column
Written consent was obtained from each volunteer while lansoprazole enantiomers were analyzed on the
before the study. The protocol was approved by the S ˜a o chiral column using acetonitrile:water (35:65 v/v), which
Francisco University Medical School Ethics Committee was delivered by Pump 2. The flow rate used was of
and is in accordance with the Declaration of Helsinki.
1.0 ml/min and the absorbance was measured at 285 nm.
Analysis time was 25 min.
Equipment and Columns
Method Validation
The HPLC system consisted of two Shimadzu LC-
1
0ATVP pumps (Kyoto, Japan), with one of the pumps hav-
The following validation parameters were evaluated:
ing a FCV-10AL valve for selecting solvent, a DGU-14A selectivity, linearity, recovery, accuracy and precision,
degasser model, an autoinjector model SIL 10AVP, a SPD– limit of quantification (LOQ), limit of detection (LOD), and
1
0AVP UV–vis detector, a SPD-M10AVP diode array de- stability. The validation parameters were in conformity
1
4
tector, and a SCL 10AVP interface. A HPLC 7000 Nitronic with the internationally accepted criteria.
EA (Sulpelco, St. Louis, MO) six-port valve was used for
the automated column-switching. Data acquisition was spiked with (6)-lansoprazole, and plasma samples of the
done on a Shimadzu CLASS-VP software. volunteers were analyzed to evaluate the selectivity of the
Chromatograms of drug-free plasma samples, plasma
The chiral column of amylose tris(3,5-dimethoxyphenyl- method. Plasma samples of the volunteers were assayed
carbamate) coated (20% w/w) onto APS-Nucleosil (7 lm using a photodiode array UV–vis detector and the peak
˚
particle size and 500 A pore size) (150 mm 3 4.6 mm I.D.) purity of each enantiomer was evaluated.
and the RAM BSA-octyl column (Hypersil, 10 lm particle
size and 120 A pore size) (100 mm 3 4.6 mm I.D.) were (0.20, 0.40, 0.80, 1.2, 2.0, 2.5, 3.0, and 4.0 lg/ml) prepared
The linearity was evaluated using calibration standards
˚
Chirality DOI 10.1002/chir

ENANTIOMERIC DETERMINATION OF LANSOPRAZOLE IN HUMAN PLASMA
37
Fig. 2. Schematic diagram of the column-switching HPLC system.
in triplicate. Plasma calibration curves were constructed exceed 15% of the coefficient of variation (CV) except for
by plotting the peak area against the concentration of each the LOQ, where it should not exceed 20% of the CV.
enantiomer of lansoprazole.
Stability was evaluated for stock solutions stored and
The extraction and transfer efficiency of each enan- refrigerated at 148C for 4 mo. The chemical stability of
tiomer of lansoprazole (recovery) from human plasma by plasma samples containing (6)-lansoprazole and drug-free
RAM column was determined by analyzing the QC sam- plasma samples (matrix) were tested in the following con-
ples. The efficiencies were calculated by dividing mean ditions: (a) sitting at room temperature for 24 h (autosam-
peak areas obtained from the spiked plasma samples by pler stability) and (b) stored at 2708C for 6 mo (long-term
mean peak areas obtained from samples of the lansopra- stability). The analytes were considered stable if the varia-
zole prepared in mobile phase as the solvent.
tion of the concentrations between the assays were less
The accuracy and precision of the method were deter- than 15% of initial time response.
mined by quintuplicate analyses at the three QC samples
(
0.48, 2.4, and 3.6 lg/ml). Calibration standards and QCs
Human Study
were analyzed on three different days to determine the
intra and interday variability.
A single 30 mg (two capsules containing 15 mg) dose of
The LOQ and the LOD were measured by preparing racemic lansoprazole was administered orally to nine
spiked plasma samples with serial diluted solutions. LOD healthy volunteers after an overnight fast. Venous blood
was calculated taking a signal-to-noise ratio of 3 as criteria. samples were collected in heparinized vacutainer tubes at 0
The acceptance criteria for the accuracy was to be (predose) and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, and 8.0 h af-
within 615% of the accepted true value, except at the LOQ ter dosing. The tubes were centrifuged at 2000g for 10 min,
where 620% were accepted. The precision should not the plasma collected and stored at 2708C until analysis.
TABLE 1. Time events for the switching of columns and of mobile phases
Time (min)
Pump
Event
Valve position
0
5
9
1
1
1
.00–5.00
.01–9.59
.60–12.50
2.51–25.00
4.01–19.00
9.01–25.00
Pump 1 (eluent A)
Pump 1 (eluent B)
Pump 1 (eluent B)
Pump 2 (eluent D)
Pump 1 (eluent C)
Pump 1 (eluent A)
Plasma proteins are excluded by RAM column
Elution of retained components on the RAM
Analytes are transferred to the chiral column
Analysis of the lansoprazole enantiomers
Washing of RAM column
1
1
2
1
1
1
Conditioning of RAM column
Eluents used in Pump 1: (A) H
Eluent used in Pump 2: (D) CH
2
O, (B) CH
3 2 3
CN:H O (35:65 v/v), (C) CH CN.
3
CN:H O (35:65 v/v). Flow rate: 1.0 ml/min, k: 285 nm.
2
Chirality DOI 10.1002/chir

3
8
GOMES ET AL.
lose and amylose tris(3,5-dimethylphenylcarbamate)
phases and amylose tris[(S)-1-phenylethylcarbamate] and
RESULTS AND DISCUSSION
Method Development
tris(3,5-dimethoxyphenylcarbamate) phases, on multimo-
Sample preparation is often considered as the most vital
19
dal elution, was carried out. Great differences in enantio-
step in HPLC analysis in the bioanalytical field. Proteins
present in the matrix can precipitate or denature and
adsorb onto the packing material, leading to back-pressure
build-up, changes in retention time, and decreased column
efficiency. Some of the most commonly used sample prep-
aration techniques include liquid–liquid extraction, protein
precipitation, and solid-phase extraction. However, these
methods increase the total analysis time and reduce the
recovery of the analytes of interest. To eliminate the prob-
lems such as coprecipitation of the analytes with proteins
and loss of analytes during extraction and to avoid the
adsorption of protein onto the analytical column, a large
number of different restricted-access media (RAM) sup-
ports have been developed to allow the direct injection of
selectivity for each phase were observed on the different
modes of elution for the series of benzimidazoles investi-
1
5,16
biological fluid samples into HPLC system.
These col-
umns possess the ability of excluding macromolecules,
whereas analytes are, generally, retained by hydrophobic
interactions.
Cass and collaborators have reported on the preparation
and application of a RAM BSA column coupled to a chiral
polysaccharide column phase for the analysis of pantopra-
1
7,18
zole and omeprazole enantiomers
and for the determi-
1
3
nation of metyrapol enantiomers and metyrapone by
achiral–chiral chromatography in human plasma.
The RAM BSA column can be characterized by a hydro-
philic outer phase and a hydrophobic inner phase. Thus,
large molecules such as proteins are excluded in the void
volume whereas the small hydrophobic analytes are selec-
tively retained.
The column-switching system used for the coupling of
the RAM BSA and the chiral columns is the one showed
schematically in Figure 2.
The role of the RAM column in this column-switching
system is to remove the proteins and fractionate a zone
containing the analyte. To determine the elution profile
and retention times of the analyte in the plasma matrix,
the RAM column was directly connected to UV detector.
The plasma samples were applied when the valve was in
Position 1. Water, delivered by Pump 1, eluted mainly the
proteins of the sample from the RAM column to waste
whereas the analytes were retained on the hydrophobic
phase of the sorbent. Five minutes after the sample injec-
tion, acetonitrile:water (35:65 v/v) was applied for compres-
sion of the lansoprazole chromatographic band, and the
switching valve was rotated to Position 2, coupling the
RAM column to the chiral column. The switching time was
set from 9.60 to 12.5 min to transfer the lansoprazole to the
analytical column. The enantioselective analyses were per-
formed using acetonitrile:water (35:65 v/v), which was
delivered by Pump 2, at a flow rate of 1.0 ml/min (Position
1
; Fig. 2). The RAM column was first cleaned with 100%
acetonitrile, and then, it was conditioned with water by
Pump 1 while the separation was carried out on the amy-
lose tris(3,5-dimethoxyphenylcarbamate) chiral column.
Previously, a complete study for the enantioresolution of
Fig. 3. Typical chromatograms of (a) plasma sample from one volun-
teer collected 1.5 h after an oral dose of 30 mg of (6)-lansoprazole show-
ing the time-window used for transferring the (6)-lansoprazole from the
RAM to the chiral column, (b) drug-free plasma, and (c) spiked plasma
omeprazole, lansoprazole, and pantoprazole using cellu- with (6)-lansoprazole 0.10 lg/ml.
Chirality DOI 10.1002/chir

ENANTIOMERIC DETERMINATION OF LANSOPRAZOLE IN HUMAN PLASMA
39
TABLE 2. Extraction efficiencies of the lansoprazole
both enantiomers were efficiently performed by RAM
column.
enantiomers from human plasma (n 5 5)
The intra and interday accuracy and precision of the
method were determined for lansoprazole enantiomers by
analyzing five replicates of the three QC plasma samples
representing the entire range of the calibration curves
Recovery (%)
Concentration
added (lg/ml)
(1)-Lansoprazole
(2)-Lansoprazole
0
1
1
.24
.2
.8
90.2
92.7
89.6
98.7
94.8
90.7
(low, medium, and high concentrations) on three different
days. The accuracies were evaluated by back-calculation
and expressed as the percent deviation between found and
added concentrations of each enantiomer and the preci-
sion are expressed as CV. Table 3 shows that CV was
gated. The amylose tris(3,5-dimethoxyphenylcarbamate)
phases showed excellent enantioselectivity for lansopra-
2
.40–10.0% for (1)-lansoprazole and 2.95–9.18% for (2)-lan-
soprazole at different concentrations. In addition, the accu-
racy was estimated to be within 615% for both enantio-
mers. These results suggest that the method is accurate
and reproducible for the determination of lansoprazole
enantiomers in human plasma.
The stock solutions of (6)-lansoprazole prepared in
methanol proved to be stable for at least 4 mo when stored
at 148C and no evidence of degradation of the analytes
zole (a 5 1.72 and R 5 3.57) using acetonitrile:water
s
(50:50 v/v) as solvent. Because reversed-phase conditions
are particularly adapted to bioanalytical applications, this
polysaccharide-based chiral stationary phase was selected
for the work.
Method Validation
The chromatogram of plasma sample from one volun- was observed on the chromatograms during this period.
teer collected 1.5 h after an oral dose of 30 mg of (6)-lan- The autosampler stability and the long-term storage were
soprazole showing the time-window used for transfer the evaluated. The permanence in the autosampler tray for 24
(
6)-lansoprazole from the RAM column to the chiral col- h at room temperature (ꢀ228C) had no significant effect
umn is illustrated in Figure 3a. Figures 3b and 3c shows on the quantitative determination of lansoprazole enan-
the chromatograms, respectively, of the analysis of blank tiomers in plasma samples. No sign of degradation were
plasma samples and spiked plasma with (6)-lansoprazole observed in drug-free plasma samples (matrix) and of
0
.10 lg/ml analyzed at the established conditions. No en- plasma samples containing (6)-lansoprazole stored at
dogenous compounds interfered with the detection of the 2708C for 6 mo (long-term stability).
lansoprazole enantiomers.
Plasma calibration curves were constructed by plotting
the peak area against the concentrations of each lansopra-
Application of the Method
zole enantiomer from 0.10 to 2.0 lg/ml. The following
The present method was applied for the determination
regression equations and correlation coefficients were of the enantiomers of lansoprazole in plasma samples of
obtained: y 5 259337x 2 15982 (r 5 0.9985) for the (1)- nine healthy volunteers after a 30 mg oral dose of racemic
enantiomer and y 5 259596x 2 12659 (r 5 0.9984) for the lansoprazole administration. To evaluate if the selectivity
(2)-enantiomer. The CV of each calibration standard (n 5 of the method was maintained in the presence of lansopra-
5) varied from 1.94 to 11.8% with accuracy that varied from zole metabolites, plasma samples from one volunteer col-
91.1 to 119% for the (1)-enantiomer and a CV of 1.98 to lected at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, and 8.0 h after
8.54% with accuracy of 91.9 to 116% for the (2)-enan- the dose administration were analyzed, at the established
tiomer.
conditions but using a photodiode array detector and the
The lower concentration calibration standard (0.10 lg/ peak purity of each enantiomer was examined. No interfer-
ml) was taken as the LOQ in both cases (accuracy within ing metabolites were detected on the samples examined,
620% and CV < 20%). The LOD was 0.025 lg/ml for each demonstrating the selectivity of the method.
enantiomer. After analysis of the 70th volunteer plasma sample a
The extraction and transfer efficiencies of each enan- compound, which coeluted with the (1)-lansoprazole was
tiomer of lansoprasole obtained at the three QC levels are detected. Despite this, the QC controls did not reveal this
given in Table 2. The results show that the extractions for interference. However, the presence of this interfering
TABLE 3. Accuracy and intra- and interday variability for the assay of lansoprazole enantiomers in human plasma
First day (n 5 5)
Second day (n 5 5)
Third day (n 5 5)
Pooled (n 5 15)
Concentration
Enantiomers
(lg/ml)
Accuracy (%) CV (%) Accuracy (%) CV (%) Accuracy (%) CV (%) Accuracy (%) CV (%)
(
1)-Lansoprazole
0.24
109
85.0
91.8
112
85.0
91.8
5.15
10.0
2.72
3.21
9.18
2.99
113
87.1
92.3
110
87.2
91.8
2.98
6.85
7.41
3.70
6.57
7.47
104
89.4
88.2
105
89.5
88.1
3.17
2.40
7.37
2.95
2.96
7.80
108
87.2
90.8
109
87.2
90.6
5.06
6.42
5.65
3.27
6.24
6.09
1
1
.2
.8
(2)-Lansoprazole
0.24
1
1
.2
.8
Chirality DOI 10.1002/chir

4
0
GOMES ET AL.
chromatographic band was observed subsequently during
the reanalysis of the volunteer and predose samples.
Thus, we determined that this interference resulted from
memory effect from the RAM column during analysis of
the volunteer’s sample. The mobile phase used in the chi-
ral column was altered from acetonitrile:water (35:65 v/v)
to acetonitrile:water (30:70 v/v) and the interfering band
was eliminated. Then, the method was revalidated. A
new plasma calibration curve was constructed for each
enantiomer (y 5 146290x 2 5558.6; r 5 0.9993 for the
(1)-enantiomer and y 5 144752x 2 7756.3; r 5 0.9979
for the (2)-enantiomer) and one intra-assay accuracy and
precision was determinate by quintuplicate analyses at
the three QC samples. All accuracy and precision results
led to satisfactory intraday accuracy and precision, with
CV ꢁ 5.82% and accuracy within 615% for both enantio-
mers. The analysis of the other clinical samples was per-
formed in this new condition.
Fig. 5. Chromatograms of plasma sample from one volunteer collected
.5 h after an oral dose of 30 mg of (6)-lansoprazole.
1
The same C RAM BSA and analytical chiral columns
8
were used during the method development and validation
and also for the analysis of the clinical samples collected
from nine volunteers. The chromatographic performance
of both columns was maintained during the complete
work. The upper limit of plasma samples has not yet been
determined for the RAM BSA columns.
The assay proved to be adequate for establishing phar-
macokinetic parameters in all samples evaluated.
teer collected 1.5 h after an oral dose of 30 mg of racemic
lansoprazole.
On the enantioselective pharmacokinetics of lansopra-
zole, it was reported that the plasma concentrations of the
1
3,17,18,20
(
1)-lansoprazole after oral administration of racemic lanso-
2
1
7
4,8
prazole to rats, dogs, and humans were higher than
those of the (2)-lansoprazole. In these works, the authors
reported that the disposition of the enantiomers of lanso-
prazole appears to be influenced by enantioselective
protein binding and by enantioselective metabolism of lan-
Disposition of Lansoprazole Enantiomers
in Healthy Subjects
In this study, the plasma concentrations of the (1)-enan- soprazole in liver microsomes.
tiomer were consistently higher than those of the (2)-enan-
The (1)-lansoprazole, which is more extensively bound
tiomer in all plasma samples obtained from nine subjects to serum proteins, is expected to be poorly distributed and
after oral administration of 30 mg racemic lansoprazole.
slowly metabolized, resulting in higher serum concentra-
The mean 6 SD plasma concentration-time profile of tions than those of (2)-lansoprazole. Consequently, enan-
both enantiomers in the plasma samples of the nine volun- tioselective protein binding might partially influence the
teers are shown in Figure 4 and the chromatogram at Fig- enantioselective disposition of lansoprazole after oral
7
,8
ure 5 shows the analysis of one sample of the same volun- administration.
The results of the work reported here is in agreement
8
with previously observed results of lansoprazole showing
higher concentration of (1)-lansoprazole in the plasma
samples of all nine volunteers in the study. Thus, the use
of lansoprazole was not able to discriminate the volunteers
as extensive or poor metabolizers as happened when pan-
1
7
18
toprazole and omeprazole were given to this same vol-
unteer group.
CONCLUSIONS
A rapid, selective, and precise HPLC method based on
the direct injection of biological samples was developed
and validated for the determination of the enantiomers of
lansoprazole in human plasma. The HPLC method with
automated column-switching and UV detection was per-
formed by coupling a C RAM BSA column to a chiral
8
polysaccharide column. The total analysis time was only
2
5 min per sample. The RAM BSA column had excellent
Fig. 4. Mean 6 SD plasma concentration-time profiles of (1)-lansopra-
zole and (2)-lansoprazole after a single oral dose of 30 mg of (6)-lanso-
prazole.
lifetime and at least 350 plasma samples of 100 ll each
were analyzed.
Chirality DOI 10.1002/chir

ENANTIOMERIC DETERMINATION OF LANSOPRAZOLE IN HUMAN PLASMA
41
The method was able to quantify the lansoprazole enan- 10. Belaz KRA, Coimbra M, Barreiro JC, Montanari CA, Cass QB. Multi-
milligram enantioresolution of sulfoxide proton pump inhibitors by liq-
uid chromatography on polysaccharide-based chiral stationary phase.
J Pharm Biomed Anal 2008;47:81–87.
tiomers in a series of nine Brazilian healthy volunteers
who received an oral dose of 30 mg of racemic lansopra-
zole. The main advantage of this new method over previ-
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cleanup and determination in a completely automatized
system.
12. Matlin SA, Tiritan ME, Crawford AJ, Cass QB, Boyd DR. HPLC with
carbohydrate carbamate chiral phases—influence of chiral phase-
structure on enantioselectivity. Chirality 1994;6:135–140.
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Chirality DOI 10.1002/chir