application fungal biotransformatiostudies

7/27/2019 Application Fungal Biotransformatiostudies

1/11

ORIGINAL PAPER

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepeziland 6-O-desmethyl donepezil in culture medium: applicationto fungal biotransformation studies

Thiago Barth &Raphael Conti &Mnica Tallarico Pupo &

Laura Tiemi Okano &Pierina Sueli Bonato

Received: 12 April 2012 /Revised: 4 May 2012 /Accepted: 7 May 2012 /Published online: 29 May 2012# Springer-Verlag 2012

Abstract An high performance liquid chromatography

(HPLC) method for the enantioselective determination ofdonepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and

6-O-desmethyl donepezil (6-ODD) in Czapek culture medi-

um to be applied to biotransformation studies with fungi is

described for the first time. The HPLC analysis was carried

out using a Chiralpak AD-H column with hexane/ethanol/

methanol (75:20:5,v/v/v) plus 0.3 % triethylamine as mobile

phase and UV detection at 270 nm. Sample preparation was

carried out by liquidliquid extraction using ethyl acetate as

extractor solvent. The method was linear over the concen-

tration range of 10010,000 ng mL1 for each enantiomer of

DPZ (r0.9985) and of 1005,000 ng mL1 for each enan-

tiomer of 5-ODD (r0.9977) and 6-ODD ( r0.9951).

Within-day and between-day precision and accuracy evalu-

ated by relative standard deviations and relative errors,

respectively, were lower than 15 % for all analytes. The

validated method was used to assess DPZ biotransformation

by the fungi Beauveria bassiana American Type Culture

Collection (ATCC) 7159 andCunninghamella elegansATCC

10028B. Using the fungus B. bassiana ATCC 7159, a

predominant formation of (R)-5-ODD was observed whilefor the fungus C. elegans ATCC 10028B, DPZ was bio-

transformed to (R)-6-ODD with an enantiomeric excess

of 100 %.

Keywords Beauveria bassiana . Cunninghamella elegans .

Donepezil . Enantioseparation . 5-O-Desmethyl donepezil .

6-O-Desmethyl donepezil

Introduction

Donepezil (DPZ) (Fig. 1a), 2,3-dihydro-5,6-dimethoxy2-

[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-one

[1], is a reversible and selective inhibitor of acetylcholines-

terase [2], therapeutically used for the treatment of Alz-

heimers disease [3]. DPZ is marketed as a racemic

mixture [4] and its enantiomers have different extents of

inhibition against acetylcholinesterase in vivo and in vitro

[5]. However, no information is available concerning the most

active enantiomer.

An in vivo study carried out in rats showed thatO-deme-

thylation, N-dealkylation, andN-oxidation are the main reac-

tions involved in the biotransformation of DPZ. In addition,

this study showed the predominant formation of the metabo-

lite 6-O-desmethyl donepezil (6-ODD) (Fig. 1b)[6]. The in

vivo metabolism study of DPZ in humans suggests that the

isoenzymes involved in its metabolism are CYP2D6 and

CYP3A4. The major metabolites observed were 6-ODD, 5-

O-desmethyl donepezil (5-ODD) (Fig.1c), DPZ-cis-N-oxide,

and the glucuronide forms of 6-ODD and 5-ODD [7]. The

6-ODD is the only metabolite that has pharmacological activ-

ity similar to DPZ [8]. No information is available concerning

the enantioselective metabolism of this drug.

T. Barth (*) : P. S. Bonato (*)

Departamento de Fsica e Qumica, Faculdade de Cincias

Farmacuticas de Ribeiro Preto, Universidade de So Paulo,

Av. Caf SN,

Ribeiro Preto, SP 14040-903, Brazile-mail: [email protected]

e-mail: [email protected]

R. Conti : M. T. Pupo

Departamento de Cincias Farmacuticas, Faculdade de Cincias

Farmacuticas de Ribeiro Preto, Universidade de So Paulo,

Ribeiro Preto, SP 14040-903, Brazil

L. T. Okano

Departamento de Qumica, Faculdade de Filosofia,

Cincias e Letras de Ribeiro Preto, Universidade de So Paulo,

Ribeiro Preto, SP 14040-901, Brazil

Anal Bioanal Chem (2012) 404:257266

DOI 10.1007/s00216-012-6107-3
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


2/11

The enantioselective determination of DPZ in biologicalmatrices is described in only few papers. In 1992, Haginaka

and Seyama [9] reported the resolution of DPZ enantiomers

in rat plasma by HPLC using a protein-based stationary

phase (ovomucoid, Ultron ES-OVM) and protein precipita-

tion for sample preparation. Matsui et al. [10] developed an

HPLC method for the enantioselective determination of

DPZ enantiomers in dog plasma with a protein-based sta-

tionary phase (avidin, Bioptick AV-1) and direct plasma

injection by column-switching procedure. In 1999, the same

group [5] reported another HPLC method for the enantiose-

lective determination of DPZ enantiomers in human plasma

using the same protein-based stationary phase and liquid

liquid extraction for sample preparation. In turn, Radwan et

al. [4] reported a method for the enantioselective determina-

tion of DPZ in rat plasma and pharmaceutical formulations

using liquid-liquid extraction and HPLC with polysaccharide-

based stationary phase (cellulose tris(3,5-dimethylphenylcar-

bamate)). More recently, DPZ enantiomers were determined

in rabbit plasma by capillary electrophoresis using sulphated-

-cyclodextrin as chiral selector and liquid-liquid extraction

for sample preparation [11]. Up to now, the simultaneous

enantioselective determination of DPZ, 5-ODD, and 6-ODD

in biological samples has not been reported.

The pharmacokinetic, pharmacodynamics, and toxico-

logical enantioselective properties of DPZ and especially

of its metabolites have been little studied. To perform these

studies, milligrams of metabolites in the enantiomerically

pure form are required. A strategy to obtain them is the use

of fungi in biotransformation processes [12]. The synthesis

of optically active compounds by using microbial models

offers advantages compared with chemical synthesis, be-

cause it can be highly enantiomeric and regio-selective

under mild conditions [13]. Biotransformation studies with

fungi may also provide information for further correlationswith enantioselective biotransformation in vivo, for simulat-

ing the mammalian metabolism [14, 15]. These considera-

tions justify the interest in developing an enantioselective

method for the simultaneous determination of DPZ, 6-ODD,

and 5-ODD in culture medium to be used in biotransformation

studies of DPZ by fungi.

Experimental

Chemicals and reagents

The reference substancesrac-donepezil hydrochloride,rac-

5-ODD, and rac-6-ODD were purchased from Toronto Re-

search Chemicals (North York, ON, Canada). Methanol and

ethanol were purchased from JT Baker (Phillipsburg, NJ,

USA), 2-propanol was obtained from Fisher Scientific (Fair

Lawn, NJ, USA) and hexane (n-hexane 95 %) was pur-

chased from Tedia (Fairfield, OH, USA), all of chromato-

graphic grade. Triethylamine was obtained from JT Baker

(Phillipsburg, NJ, USA). Ethyl acetate was obtained from

Tedia (Fairfield, OH, USA). The boric acid and potassium

dihydrogenphosphate were obtained from Merck (Darmstadt,

Germany), and sodium tetraborate decahydrate was purchasedfrom JT Baker (Phillipsburg, NJ, USA). All these chemicals

were of analytical grade in the highest purity available. Water

was purified with a Milli-Q plus system (Millipore, Bedford,

MA, USA).

Reference substance solutions

The DPZ, 5-ODD, and 6-ODD stock solutions were pre-

pared at the concentration of 1 mg mL1. The working

Fig. 1 Chemical structures of

DPZ (a), 6-ODD (b), and

5-ODD (c). * Represent the

chiral center

258 T. Barth et al.
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


3/11

solutions ofrac-DPZ were prepared at the concentrations of

4, 12, 40, 120, 300, and 400 g mL1 while therac-5-ODD

and rac-6-ODD solutions were 4, 12, 30, 60, 150, and

200 g mL1. All of them were prepared in methanol on a

free-base basis. The solutions were stored frozen at20 C

and protected from light.

Liquid chromatographic conditions

The liquid chromatographic analyses were conducted using

a Shimadzu chromatograph (Kyoto, Japan), equipped with

an LC-10AS solvent pump unit, a SPD-10A UVvis detec-

tor operating at 270 nm. The system control was carried out

by a SCL-10A VP controller. Injections were performed

manually through a 50-L loop with a Rheodyne model

7125 injector (Cotati, CA, USA). A Shimadzu LC solution

software, version 1.22 SP1, was used for system control and

data acquisition. The resolution of DPZ, 5-ODD, and 6-ODD

enantiomers was carried out at room temperature (25 2 C)

on a Chiralpak AD-H column (1504.6 mm, 5 m par-ticle size, Chiral Technologies, Exton, PA, USA) using

hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % trie-

thylamine as the mobile phase at a flow rate of 1.5 mL min1.

A CN guard column (44 mm, 5-m particle size, Merck,

Darmstadt, Germany) was used to protect the analytical

column.

Elution order determination

The enantiomers of DPZ, 5-ODD and 6-ODD were obtained

by semi-preparative analysis of the racemates under the

HPLC conditions described in the present paper (see Liquid

chromatographic conditions). Furthermore, the collected

enantiomers of DPZ were analyzed on a Chiralcel OD

column (25 4.6 mm, 10 m particle size) using hex-

ane/2-propanol/TEA (87:12.9:0.1, v/v/v) as mobile phase,

according to the conditions described by Radwan et al. [4].

Then, the retention times of the enantiomers from both

studies were compared and the elution order established.

In turn, the elution order of 5-ODD and 6-ODD enan-

tiomers was determined by obtaining the circular dichro-

ism (CD) spectra of the pure enantiomers of DPZ, 5-ODD,

and 6-ODD (200 to 400 nm) on a JASCO J-810 spectropo-

larimeter equipped with the temperature control apparatus

JASCO PTC-423S, using a quartz cuvette with a 1.0-cm

optical path at 25 C (Easton, MD, USA). Subtractions of

base line (mobile phase) were carried out for all analysis. The

standard scanning conditions were at a rate of 200 nm min1

continuous mode, spectral bandwidth 1 nm, and a response of

0.5 s. The 5-ODD and 6-ODD enantiomer that pre-

sented the same cotton effect of (R)-DPZ was considered

(R) and the one that presented the same cotton effect of (S)-

DPZ was considered (S).

Extraction procedure

DPZ, 5-ODD, and 6-ODD were extracted from the Czapek

culture medium by a liquid-liquid extraction procedure.

Aliquots of 0.5 mL Czapek medium spiked with 25 L of

standard solutions of DPZ, 5-ODD, and 6-ODD or samples

obtained in the biotransformation process were transferred

to 10 mL glass tubes, and buffered with 0.5 mL of sodiumborate buffer 0.1 mol L1, pH 9. The samples were mixed by

vortex agitation for 20 s. Then, a 4-mL aliquot of extraction

solvent, ethyl acetate, was added. The tubes were shacked

for 15 min using a Vibrax VXR agitator (IKA, Staufen,

Germany) set at 1,500 rpm and then centrifuged at

1,800gfor 5 min, at 4 C. The organic layers (3 mL) were

transferred to 10 mL conical glass tubes. The solvent was

evaporated to dryness under a stream of compressed air at

room temperature. The residues were dissolved in 100 L of

mobile phase and vortex mixed for 20 s, and then 50 L

were analyzed by the HPLC system.

Racemization assessment during extraction

and biotransformation procedures

DPZ may racemize, via a keto-enol intermediate since the

chiral center is adjacent to a carbonyl group [5,9,10]. So,

the influence of the biotransformation and extraction con-

ditions on the racemization of DPZ, 5-ODD and 6-ODD

was evaluated. Firstly, the enantiomers of DPZ, 5-ODD and

6-ODD were obtained by semi-preparative analysis of the

racemates under the HPLC conditions described in the pres-

ent paper (see Liquid chromatographic conditions). After

separation, the fractions containing each enantiomer were

evaporated to dryness under a stream of compressed air and

the residues were dissolved in methanol to obtain a final

concentration of approximately 50 g mL1. The enantio-

meric excess (ee) of these solutions was determined by the

equation ee0(AB/A+B) 100, whereA is the peak area of

the enantiomer in higher concentration and B is the peak

area of the enantiomer in lower concentration [16].

To determine the racemization in the biotransformation

conditions, 1-mL aliquots of DPZ, 5-ODD, and 6-ODD

enantiomers solutions were added to Erlenmeyer flasks con-

taining 100 mL of Czapek culture medium and submitted to

the same conditions used in the biotransformation procedure

(see Donepezil biotransformation procedure). Daily, dur-

ing the period of biotransformation (7 days), aliquots of

1 mL (n03) were collected. At the end of the biotransforma-

tion study, the samples were extracted and analyzed in tripli-

cate and the ee was determined. This ee values were compared

with theee obtained after the enantiomer separation.

To evaluate the influence of sample pH during the ex-

traction procedure on the racemization, samples of 0.5 mL

of Czapek culture medium were spiked with 25 L of the

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil 259
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


4/11

solutions of each enantiomer of DPZ, 5-ODD, and 6-ODD

(n03). After that the pH of the samples were adjusted by the

addition of 0.5 mL of buffer solutions and the extraction was

performed. The evaluated pH value s were 7.0 (sodium

phosphate buffer, 0.1 mol L1), 9.0 (sodium borate buffer,

0.1 mol L1), and 10.0 (sodium borate buffer, 0.1 mol L1).

The ee of the enantiomer solutions obtained after enantio-

mer separation was also used for comparison with the eedetermined during the evaluation of the pH effect on the

racemization.

Method validation

To determine the extraction recovery, aliquots of Czapek

culture medium (0.5 mL) were spiked with DPZ at the

concentrations of 300, 3,000, and 7,500 ng mL1 for each

enantiomer (n03) and 300, 1,500, and 3,750 ng mL1 for

each enantiomer of 5-ODD and 6-ODD and submitted to the

extraction procedure. Another set of samples were prepared

extracting 0.5 mL aliquots of Czapek medium and thenspiking the extract with the same amounts of DPZ, 5-

ODD, and 6-ODD enantiomers. The recovery was deter-

mined by the ratio of the areas of the samples with analytes

extracted and non-extracted and expressed as percentage of

the amount extracted.

Calibration curves were obtained by spiking 0.5 mL aliquots

of Czapek culture medium with 25L of standard solutions of

rac-DPZ, in the concentration range of 4400 g mL1,

resulting in concentrations of 10010,000 ng mL1 for

each enantiomer. The calibration curves for rac-5-ODD

and rac-6-ODD were prepared similarly at the concen-

tration range of 1005000 ng mL1 for each enantiomer.

The linearity of the calibration curves was determined

using the correlation coefficient (r) and the F test for lack-

of-fit (FLOF) using a p value of 0.05. MINITAB Release

version 14.1 (State College, PA, USA) was used to perform

the statistical calculations.

The sensitivity of the method was evaluated by determin-

ing the quantification limit (LOQ). The LOQ was defined as

the lowest enantiomer concentration that could be deter-

mined with accuracy and precision below 20 % [17] over

five analytical runs. The LOQ was determined by using

aliquots of Czapek culture medium (0.5 mL) spiked with

100 ng mL1 of each enantiomer (the lowest point in the

calibration curve).

The precision and accuracy of the method were eval-

uated by within-day (n05) and between-day (n03)

assays using Czapek culture medium spiked with DPZ

at the concentrations of 300, 3,000, and 7,500 ng mL1

for each enantiomer and 300, 1,500, and 3,750 ng mL1 for

each enantiomer of 5-ODD and 6-ODD. The results obtained

were expressed as relative standard deviation (RSD, %) and

relative error (RE (%)).

Freeze-thaw cycle stability, short-term room temperature

stability and stability in the biotransformation conditions

were determined. To perform the freezethaw cycle stability,

three aliquots (n03) of samples prepared in Czapek culture

medium at the concentration of 300 and 7,500 ng mL1 of

each enantiomer of DPZ and 300 and 3,750 ng mL1 of each

enantiomer of 5-ODD and 6-ODD were stored at20 C for

24 h and thawed at room temperature. When completelythawed, the samples were refrozen for 12 h under the same

conditions. The freezethaw cycle was repeated twice, and

then the samples were analyzed on the third cycle. For the

determination of short-term room temperature stability, ali-

quots of samples prepared in Czapek culture medium at the

concentrations specified above were kept at room tempera-

ture (222 C) for 12 h and analyzed. To determine the

stability in the biotransformation conditions, an aliquot of

2 mg of rac-DPZ (free-base basis) dissolved in 1 mL of

sterile water was added to Erlenmeyer flasks containing

100 mL of Czapek medium (10 g mL1 of each enantio-

mer), and submitted to the same conditions used in thebiotransformation procedure (see Donepezil biotransfor-

mation procedure). Daily, during the period of biotransfor-

mation (7 days), aliquots of 0.5 mL (n03) were analyzed.

The peak areas obtained from the stability studies were

compared with the peak areas obtained with freshly pre-

pared samples at the same concentration and were consid-

ered stable if the deviation (expressed as RE (%)) from the

fresh samples was within15 %.

The selectivity of the method was evaluated by analyzing

sterile Czapek medium and sterile Czapek medium added of

fungal mycelium under the conditions previously estab-

lished (see Donepezil biotransformation procedure).

Fungi

The fungiBeauveria bassianaAmerican Type Culture Col-

lection (ATCC) 7159 and Cunninghamella elegans ATCC

10028B, purchased from the ATCC (Manassas, VA, USA)

were used in this biotransformation study.

Donepezil biotransformation procedure

Three discs of 0.5 cm of diameter (potato dextrose agar

plugs) containing the fungal mycelia were aseptically trans-

ferred to 9.0-cm diameter Petri dishes containing PDA me-

dium (potato, dextrose, and agar) and allowed to grow for

6 days at 30 C. Biotransformation was performed using a

two-stage fermentation protocol [18, 19]. In the first stage

(pre-culture), three 0.5-cm uniform discs were cut with a

transfer tube (Fischer Scientific, Pittsburgh, PA, USA) and

then inoculated in 50-mL Falcon tubes containing 10 mL of

pre-fermentative liquid broth (glucose, 10 g; tryptone soy

broth, 5 g; yeast extract, 3 g; and malt extract, 10 g; per litre

260 T. Barth et al.
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


5/11

and pH adjusted to 6.2 with 0.1 mol L1 HCl solution). The

Falcon tubes were incubated for 4 days (96 h) at 30 C on a

rotatory shaker (New Brunswick Scientific Co., Inc., model

INNOVA 4300, Edison, NJ, USA) operating at 120 rpm.

In the second stage (biotransformation), the resulting myce-

lium was transferred into 250-mL Erlenmeyer flasks con-

taining 100 mL of Czapek medium [20] adjusted to pH 5.0

with a 1.0 mol L1 HCl solution.Moreover, 2 mg of DPZ (free-base basis) dissolved in

1 mL of sterile water was added to the flask. Control flasks

consisted of culture broth (Czapek) without DPZ and fungi,

sterile broth with fungal mycelium, and sterile broth with

DPZ (stability in biotransformation conditions). Biotrans-

formation experiments were carried out at 30 C, with shaking

at 120 rpm for 168 h. Aliquots of 0.5 mL were collected from

the culture flasks, submitted to extraction procedure and ana-

lyzed by HPLC.

The biotransformation kinetic studies were presented as

concentration versus collecting interval (hours) profiles. In

addition, the results obtained in the biotransformation processwere also expressed as ee. The efficiency of the biotransfor-

mation process was calculated (in percentage). It was per-

formed quantifying the amount of the 5-ODD and 6-ODD

metabolites in the culture medium and correlating this amount

with the initial amount of DPZ (time 0). The biotransforma-

tion procedure was performed in replicate (n02).

Results and discussion

Selection of the separation conditions

The chiral resolution of DPZ, 5-ODD, and 6-ODD was per-

formed using a Chiralpak AD-H column, under normal elu-

tion mode. The mobile phase was prepared using hexane/

2-propanol, hexane/2-propanol/methanol, hexane/ethanol, or

hexane/ethanol/methanol mixtures. Methanol was used to

reduce the analysis time and to improve the peak efficiency.

Triethylamine was added to these mobile phases in order to

reduce the interaction of the drugs with the silanol groups of

the silica support. Moreover, methanol and triethylamine were

important for the separation of (S)-DPZ (peak 1) and (S)-

5-ODD (peak 2) (Fig.2a). The best separation was achieved

with a mobile phase consisting of hexane/ethanol/methanol

(75:20:5, v/v/v) plus 0.3 % triethylamine, at a flow rate of

1.5 mL min1 and detection at 270 nm. Under these condi-

tions, the separation of all compounds was performed in

19 min with enough resolution (Rs1.56) (Fig.2a).

Elution order determination

The elution order for DPZ enantiomers on the Chiralpak

AD-H column was reversal than the observed by Radwan et

al. [4], which used a Chiralcel OD column. The chiral

selector for the Chiralpak AD-H and Chiralcel OD columns

are amylose tris (3,5-dimethylphenylcarbamate) and cellu-

lose tris(3,5-dimethylphenylcarbamate), respectively. So the

difference between them is the polysaccharides used to

prepare the derivative. The reversal of elution order by

changing the polysaccharide is well documented [2124].

Peak 1 corresponded to (S)-DPZ and peak 4 to (R)-DPZ

(Fig.2a).

The chiral resolution and elution order of 5-ODD and 6-

ODD enantiomers is not available in the literature. There-

fore, to determine the elution order of DPZ metabolites, a

combination of chromatographic and CD methods was used

[25, 26]. Hence, the CD spectra of 5-ODD and 6-ODD

enantiomers were compared with the CD spectra of DPZ

enantiomers. For 5-ODD and 6-ODD, the first eluted enan-

tiomers showed cotton effect similar to the (S)-DPZ and

they were considered as (S)-5-ODD (peak 2) and (S)-6-

ODD (peak 3); the second eluted enantiomers of the metab-

olites showed cotton effect similar to the (R)-DPZ and they

were considered as (R)-5-ODD (peak 5) and (R)-6-ODD

(peak 6) (Fig. 2a). The CD spectra used for the determina-

tion of the elution order are presented in Fig. 3.

Method validation

Several solvents or mixtures of them were evaluated to

extract DPZ, 5-ODD, and 6-ODD enantiomers from Czapek

Fig. 2 (a) Representative chromatogram of blank Czapek culture

medium spiked with 3,000 ng mL1 of each DPZ enantiomer and

1,500 ng mL1 of each 5- and 6-ODD enantiomers. (b) Representative

chromatogram of blank Czapek culture medium. (c) Representative

chromatogram of Czapek culture medium incubated with the fungusC.

elegans ATCC 10028B during 7 days. (d) Representative chromatogramof Czapek culture medium incubated with the fungusB. bassianaATCC

7159 during 7 days. (1) (S)-DPZ, (2) (S)-5-ODD, (3) (S)-6-ODD, (4) (R)-

DPZ, (5) (R)-5-ODD, and (6) (R)-6-ODD. Chromatographic conditions

are described in Liquid chromatographic conditions

http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


6/11

culture medium using liquid-liquid extraction. Finally the

selected solvent was ethyl acetate. DPZ and its metabolites

are basic compounds and, moreover DPZ may racemize in

aqueous solution [5,9,10]. Thus, to obtain the best recovery

in the absence of racemization, the extraction of DPZ, 5-ODD

and 6-ODD, as well as the isolated enantiomers, was evaluat-

ed in pH 7.0, 9.0, and 10.

The best recovery results were observed at pH 10, but

racemization was observed under this pH that prohibitsits use. In turn, at pH 7 and 9, the racemization was

negligible, but the recovery value was higher at pH 9, so this

pH was selected for further validation of the method

(Table 1).

The recoveries of DPZ, 5-ODD and 6-ODD were approx-

imately 97, 95, and 92 % for both enantiomers, respectively.

The RSD (%) for the recoveries of all analytes were lower

than 10.1 % (Table2).

Linear regression analyses were performed by plotting

the peak area of the enantiomer (y) versus theoretical enan-

tiomer concentrations (x). The method proved to be linear

over the concentration range of 10010,000 ng mL1 for

each DPZ enantiomer, with correlation coefficient (r)

0.9985 (Table2) and over the concentration range of 100

5,000 ng mL1 for each 5-ODD and 6-ODD enantiomer,

with correlation coefficient (r)0.9951. The linearity of the

method was also confirmed by the lack-of-fit test (Table2).

Fig. 3 CD spectra for the DPZ

(a), 5-ODD (b), and 6-ODD

(c) enantiomers

Table 1 Influence of the biotransformation process on the racemization and sample pH on the recovery and racemization

Analytes (n03)a ee initialb pH 7.0 pH 9.0 pH 10.0 Biotransformationc

ee (%) Recovery (%) ee (%) Recovery (%) ee (%) Recovery (%) ee (%)

(S)-DPZ 99.30.3 99.10.4 71.0 4.4 98.80.9 95.24.4 90.21.1 96.55.3 96.50.1

(R)-DPZ 98.70.4 98.21.1 72.04.8 97.80.7 95.94.3 89.71.3 97.15.7 97.00.2

(S)-5-ODD 98.30.8 97.9 0.7 65.36.8 97.3 0.2 93.16.2 88.5 0.9 94.2 6.7 95.30.2

(R)-5-ODD 99.30.7 98.9 0.4 63.36.6 98.2 0.5 92.56.4 89.3 0.8 93.96.8 95.50.6

(S)-6-ODD 98.70.2 98.5 0.6 55.1 8.3 97.9 0.6 89.17.8 90.3 1.6 92.17.9 97.40.5

(R)-6-ODD 99.30.3 98.7 0.5 56.7 9.9 98.0 0.7 90.28.2 91.8 1.5 93.48.3 97.60.7

DPZdonepezil, 6-ODD6-O-desmethyl-donepezil, 5-ODD5-O-desmethyl-donepezilaNumber of determinationsb Determined after enantiomer purificationc Determined after seven biotransformation days

262 T. Barth et al.
http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


7/11

The lowest concentration quantified (LOQs) by the vali-

dated method was 100 ng mL1 for all analytes. The RSD (%)

and RE (%) were lower than 20 % (Table 3).

The precision and accuracy of the method were evaluatedby within and between-day assays. The RSDs and relative

errors were lower than 15 % (Table4).

The freezethaw and short-term room temperature stabil-

ity of DPZ, 5-ODD, and 6-ODD enantiomers in Czapek

culture medium were evaluated by comparison of the peak

areas of the stability test samples with the peak areas from

freshly prepared samples and showed RSDs and relative

errors lower than 15 % (Table5). Moreover, DPZ was stable

during 168 h under the biotransformation conditions (RSD

and RE


8/11

Table 4 Precision and accuracy of the method

Analyte Nominal Concentration

(ng mL1)

Within day (n05)a Between day (n03)b

Concentration (ng mL1) RSD (%) RE (%) Concentration (ng mL1) RSD (%) RE (%)

(S)-DPZ 300 304.0 3.1 1.3 301.2 2.0 0.4

3,000 3,028.1 3.2 0.9 3,005.2 1.3 0.2

7,500 7,145.0 1.7

4.7 7,055.9 2.1

5.9(R)-DPZ 300 308.1 7.6 2.7 308.8 2.9 2.9

3,000 3,068.5 3.0 2.3 2,992.9 2.3 0.2

7,500 7,252.9 2.9 3.3 7,032.1 3.1 6.2

(S)-5-ODD 300 306.6 8.6 2.2 293.0 4.7 2.3

1,500 1,588.4 2.0 5.9 1,503.9 6.7 0.3

3,750 3,840.2 4.5 2.4 3,612.3 6.5 3.7

(R)-5-ODD 300 294.6 8.9 1.8 293.0 1.9 2.3

1,500 1,596.6 4.6 6.4 1,509.2 6.6 0.6

3,750 3,847.1 3.6 2.6 3,621.7 6.9 3.4

(S)-6-ODD 300 310.1 3.6 3.4 304.1 4.8 1.4

1,500 1,578.7 2.0 5.2 1,510.6 5.4 0.7

3,750 3,465.7 2.9

7.6 3,452.1 2.4

7.9

(R)-6-ODD 300 284.8 4.6 5.1 280.9 3.5 6.3

1,500 1,612.7 2.9 7.5 1,529.8 5.1 2.0

3,750 3,555.3 3.1 5.2 3,514.4 3.9 6.3

DPZdonepezil, 6-ODD6-O-desmethyl-donepezil, 5-ODD5-O-desmethyl-donepezil, RSD relative standard deviation in percentage,RE relative

error in percentageaNumber of determinationsbNumber of days

Table 5 Short-term room temperature and freeze/thaw stability of analytes

Analyte

(n06)

Fresh sample

concentration

(ng mL1)

Short-term room temperature (12 h) Freeze/thaw cycles

Determined concentration

(ng mL1)

RSD

(%)

RE

(%)bDetermined concentration

(ng mL1)

RSD

(%)

RE

(%)b

(S)-DPZ 289.0 286.7 2.5 0.8 275.3 2.5 4.7

6,884.8 6,743.4 2.5 2.0 6,942.1 1.9 0.8

(R)-DPZ 270.2 269.3 4.0 0.3 259.0 3.0 4.3

6,781.7 6,677.7 2.6 1.5 6,859.2 2.2 1.1

(S)-5-ODD 304.6 294.1 5.6

3.4 276.8 3.1

9.13,317.6 3,266.7 1.3 3.1 3,342 1.6 0.9

(R)-5-ODD 280.4 280.5 4.9 0.0 261.6 9.0 6.7

3,505.6 3,404.8 3.1 2.9 3,515.7 2.6 0.3

(S)-6-ODD 303.6 275.2 7.1 9.3 264.9 3.7 12.7

3,249.0 3,318.5 3.1 2.1 3,283.0 2.7 1.0

(R)-6-ODD 283.6 277.0 12.4 2.3 265.4 9.5 6.4

3,272.5 3,333.5 3.4 1.9 3,349.4 2.2 2.3

DPZdonepezil,6-ODD6-O-desmethyl-donepezil,5-ODD5-O-desmethyl-donepezil,n number of determinations,RSDrelative standard deviation

in percentage, RErelative error in percentage

264 T. Barth et al.


9/11

metabolite was also observed in the biotransformation by this

fungus. This peak can be considered a possible metabolite of

DPZ that was not studied here (peak 7, Fig. 5a).

The fungi from the genus Cunninghamella were used to

perform O-demethylation reactions in several drugs such as

muraglitazar [31], naproxen [32], pantoprazole [33], pyril-

amine [34], and verapamil [35].

The biotransformation of DPZ by the fungus C. elegans

ATCC 10028B resulted in the formation of the metabolite

(R)-6-ODD, enantioselectively. The metabolite 6-ODD is

considered the active metabolite of DPZ [8] and consists

of the predominant metabolite formed in rats [6] and

humans [7]. The formation of (R)-6-ODD was firstly ob-

served after 72 h of incubation, but the maximal concentration

of (R)-6-ODD in the culture medium, was 1,520 ng mL1

(15.1 %) in 168 h (Fig.4a). This concentration corresponds to

an ee of 100 % of (R)-6-ODD (Fig.4a), because the formation

of the enantiomer (S)-6-ODD was not observed. Therefore,

this fungus was found to be an excellent model for the pro-

duction of enantiomerically pure 6-ODD metabolite. Figure 5b

shows typical chromatogram obtained from the biotransforma-

tion process employing the fungusC. elegansATCC 10028B.

Other unidentified metabolites were also observed in the bio-

transformation by this fungus (peak 8 and 9, Fig. 5b). So the

biotransformation by the fungiB. bassiana ATCC 7159 and C.

elegansATCC 10028B deserve more studies for the isolation

and characterization of these unknown metabolites (Fig. 5a

(peak 7), b (peaks 8 and 9)).

Concluding remarks

A method for the enantioselective determination of DPZ, 5-

ODD and 6-ODD in Czapek culture medium was developed

and validated. This method is the first report for the simulta-

neous enantioselective analysis of DPZ, 5-ODD and 6-ODD,

Fig. 4 Concentration-time graphs for the biotransformation process of DPZ by fungi (a)C. elegansATCC 10028B and (b)B. bassianaATCC 7159.

Thebarsdenote the standard deviation of replicate (n02)

Fig. 5 (a) Representative chromatograms for 168-h incubation of the

fungus B. bassiana ATCC 7159 with DPZ and of Czapek culture

medium incubated with B. bassiana ATCC 7159 (fungus control)

showing that this fungus did not produce any secondary metabolite

in the retention time of the analytes. (1) (S)-DPZ, (2) (S)-5-ODD, (3)

(S)-6-ODD, (4) (R)-DPZ, (5) (R)-5-ODD, (6) (R)-6-ODD, and (7)

unknown peak. (b) Representative chromatograms for the 96 h

incubation of the fungus C. elegans ATCC 10028B with DPZ and of

Czapek culture medium incubated with C. elegans ATCC 10028B

(fungus control) showing that this fungus did not produce any second-

ary metabolite in the retention time of the analytes. (1) (S)-DPZ, (4)

(R)-DPZ, (6) (R)-6-ODD, (8, 9) unknown peaks. Chromatographic

conditions are described in Liquid chromatographic conditions

http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


10/11

and it was successfully employed to study the biotransforma-

tion of DPZ by fungi. In addition, the biotransformation study

also demonstrates, for the first time, the enantioselectivity on

the biotransformation of DPZ, since the enantioselective bio-

transformation of this drug has not been reported yet for

mammals or other biotransformation model. In this study,

the predominant formation of (R)-5-ODD by B. bassiana

ATCC 7159 with an ee of 60.6 % and (R)-6-ODD by C.elegans ATCC 10028B with an ee of 100 % were observed.

The biotransformation with the fungus C. elegans ATCC

10028B correlates with the metabolism in mammals and can

be used as an alternative to this model. Moreover, both fungi

can be also used to obtain these metabolites to be used in

toxicological and pharmacological studies.

Acknowledgments The authors are grateful to Fundao de Amparo

Pesquisa do Estado de So Paulo (FAPESP), Conselho Nacional de

Desenvolvimento Cientfico e Tecnolgico (CNPq), and Coordenao

de Aperfeioamento de Pessoal de Nvel Superior (CAPES) for finan-

cial support and for granting research fellowships.

Conflict of interest The authors have declared no conflict of interest.

References

1. Moffat AC, Osselton MD, Widdop B (2003) Clarkes analysis of

drugs and poisons, 3rd edn. Pharmaceutical Press, London

2. Geldmacher DS (2004) Expert Rev Neurother 4:516

3. Wilkinson D (2007) Psychiatry 7:914

4. Radwan MA, Abdine HH, Al-Quadeb BT, Aboul-Einein HY,

Nakashima K (2006) J Chromatogr B 830:114119

5. Matsui K, Oda Y, Nakata H, Yoshimura T (1999) J Chromatogr B

729:147155

6. Matsui K, Mishima M, Nagai Y, Yuzuriha T, Yoshimura T (1999)

Drug Metab Dispos 27:14061414

7. Tiseo PJ, Perdomo CA, Friedhoff LT (1998) Br J Clin Pharmacol

46:1924

8. Dooley M, Lamb HM (2000) Drug Aging 16:199226

9. Haginaka J, Seyama C (1992) J Chromatogr 577:95102

10. Matsui K, OdaY, OheH, TanakaS, Asakawa N (1995) J Chromatogr

A 694:209218

11. Lu YH, Zhang M, Meng Q, Zhang ZX (2006) Acta Pharm Sinic

41:471475

12. Venisetty VJ, Ciddi V (2003) Curr Pharm Biotechnol 4:123140

13. Asha S, Vidyavathi M (2009) Biotechnol Adv 27:1629

14. Smith RV, Rosazza JP (1983) J Nat Prod 46:7991

15. Azerad R (1999) Adv Biochem Eng Biotechnol 63:169218

16. Altria KD (1996) In: Shitani H, Polonsk J (eds) Quantitative

applications of the resolution of enantiomers by capillary electro-

phoresis. Blackie Academic and Professional, London

17. Guidance for Industry: Bioanalytical Method Validation (2001),United States Food and Drug Administration, Silver Spring.

Available at http://www.fda.gov/downloads/Drugs/Guidance

ComplianceRegulatoryInformation/Guidances/UCM070107.pdf.

Accessed 15 Mar 2012

18. Borges KB, Borges WS, Pupo MT, Bonato PS (2008) J Pharm

Biomed Anal 46:945952

19. Barth T, Pupo MT, Borges KB, Okano LT, Bonato PS (2010)

Electrophoresis 31:15211528

20. Alviano CS, Fabiarz SR, Travassos LR, Angluster J, Souza W

(1992) Mycopathologia 119:1723

21. Wang T, Chen YW (1999) J Chromatogr A 855:411421

22. Wang T, Chen YW, Vailaya A (2000) J Chromatogr A 902:345

355

23. Kazusaki M, Kawabata H, Matsukura H (2000) J Liq Chrom Relat

Tech 23:2819282824. Gaggeri R, Rossi D, Collina S, Manucci B, Baierl M, Juza M

(2011) J Chromatogr A 1218:54145422

25. Lmmerhofer M, Pell R, Mahut M, Richter M, Schiesel S, Zettl H,

Dittrich M, Schubert-Zsilavecz M, Lindner W (2010) J Chroma-

togr A 1217:10331040

26. Barth T, Simes RA, Pupo MT, Okano LT, Bonato PS (2011) J Sep

Sci 34:35783586

27. Grogan GJ, Holland HL (2000) J Mol Catal B: Enzym 9:132

28. Wu G, Gard A, Rosazza J (1980) J Antibiot 33:705710

29. Archelas A, Fourneron JD, Furstoss R (1988) Tetrahedron Lett

29:66116614

30. Olivo HF, Peeples TL, Ros MY, Velsquez F, Kim JW, Narang S

(2003) J Mol Catal B: Enzym 21:97105

31. Zhang D, Zhang H, Aranibar N, Hanson R, Huang Y, Cheng PT,

Wu S, Bonacorsi S, Zhu M, Swaminathan A, Humphreys G (2006)

Drug Metab Dispos 34:267280

32. Zhong DF, Sun L, Liu L, Huang HH (2003) Acta Pharm Sinic

24:442447

33. Xie ZY, Huang HH, Zhong DF (2005) Xenobiotica 35:467477

34. Hansen EB Jr, Cerniglia CE, Korfmacher WA, Miller DW, Heflich

RH (1987) Drug Metab Dispos 15:97106

35. Sun L, Huang HH, Liu L, Zhong DF (2004) Appl Environ Microbiol

70:272227277

266 T. Barth et al.
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdfhttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdfhttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdfhttp://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf


11/11

Copyright of Analytical & Bioanalytical Chemistry is the property of Springer Science & Business Media B.V.

and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright

holder's express written permission. However, users may print, download, or email articles for individual use.

application fungal biotransformatiostudies

Documents