JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES

Vol. 27, No. 3, pp. 511-519, 2004

Biotin Dissolution from Pharmaceutical

Dosage Forms Using an Automated
HPLC System
1

Hassan Y. Aboul-Enein, '* Rajaa F. Hussein,

Mahasen A. Radwan, and Sameer Al-Rawithi
3

Pharmaceutical Analysis Laboratory, Biological and Medical Research

Department, King Faisal Specialist Hospital and Research Centre,
Riyadh, Saudi Arabia
Pharmacokinetics and Therapeutic Drug Monitoring Laboratory,
Biological and Medical Research Department, King Faisal Specialist
Hospital and Research Centre, Riyadh, Saudi Arabia
Department of Clinical Pharmacy, College of Pharmacy, Science &
Medical Studies, Department for Women Students, King Saud
University, Saudi Arabia
3

ABSTRACT
A novel, rapid, accurate, and sensitive automated high-performance
liquid chromatographic assay was developed to detcamme btdtin (BI) in

""Correspondence: Hassan Y. Aboul-Enein, Pharmaceutical Analysis Laboratory,

Biological and Medical Research Department (MBC 03-65), King Faisal Specialist
Hospital and Research Center, P. O. Box 3354, Riyadh 11211, Saudi Arabia; E-mail:
enein@kfshrc.edu.sa.
511
DOI: 10.I081/JLC-120027622
Copyright 2004 by Marcel Dekkcr, Inc.

1082-6076 (Print); 1520-572X ( O n l i n e )

www.dekker.com

ORDER H W |

REPRINTS

A b o u l - E n e i n e t al.
pharmaceutical d o s a g e f o r m s and to f o l l o w i t s dissolution pattern. An
efficient separation of BI w a s performed using a stainless steel Supelcosil
L C - 1 8 c o l u m n (25 cm x 4.6 m m ; 5 u,m p a n i c l e size) preceded by a
Sentry guard column. The m o b i l e p h a s e consisted of an 8 0 % aqueous
solution (pH 2.5 adjusted with phosphoric acid) containing 2 0 %
acetonitrile delivered at a flow rate of 1 . 5 m L / m i n . T h e compound of
interest w a s detected using a photodiode array detector at 1 9 0 n m . Under
t h e s e conditions, the assay run time w a s 6 min since the retention time of
BI w a s 3.8 0 . 2 min. T h e detector response w a s linear for BI in alkaline
solution (r > 0.999) in the range of 0.01 - 2 . 0 0 p . g / m L . T h e detection and
the quantification limits for BI were 0 . 0 0 5 and 0.01 u . g / m L , respectively.
T h e dissolution data s h o w e d R S D % of 3 . 6 - 1 2 . 7 % for all BI determined
concentrations. No interferences w e r e observed from the tablet's
excipients. T h e drug content in e a c h tablet ranged from 100 to 102.5%.
T h e dissolution study o f B I O T T N * tablets revealed that B I i n U S P m e d i a
(pH 1.2) s h o w e d no dissolution up to 3 nr. H o w e v e r , a first order release
kinetic, with dissolution T s o of 14 1.3 m i n , w a s observed in U S P
media (pH7.4).
%

Key Words:

Biotin; Photodiode array detector; Dissolution; HPLC.

INTRODUCTION
B i o t i n ( B I ) , Cis h e x a h y d r o - 2 - o x o - l H - t h i e n o - [ . 3 , 4 - d ] - i m i d a z o l e - 4 - p e n t a n o i c a c i d , k n o w n a s "Vitamin H , i s a v e r y s l i g h t l y w a t e r - s o l u b l e v i t a m i n
b e l o n g i n g t o the B - c o m p l e x , w h i c h i s f o u n d i n s m a l l quantities i n all l i v i n g
cells. It exists in eight isomer forms, but only D - ( + ) - b i o t i n is biologically
active. In the intestine, biotin is absorbed through a saturable transportation
s y s t e m , w h i l e at great biotin concentrations; p a s s i v e diffusion p r e d o m i n a t e s .
L o w biotin intake has b e e n reported to result in serious biochemical disorders
in animal organisms, such as reduced carboxylase activity, inhibition of
protein and R N A synthesis, r e d u c e d antibody production, etc. T h e r e are
indications of biotin i n v o l v e m e n t in severe animal syndromes, such as the
a v i a n fatty liver and k i d n e y s y n d r o m e ( F L K S ) a n d the trout 'blue s l i m e '
disease. - - Diagnosis of biotin deficiency, as well as monitoring of biotin
l e v e l s i n b i o l o g i c a l fluids o f p a t i e n t s r e c e i v i n g b i o t i n t r e a t m e n t i s v e r y c r u c i a l .
Equally important is the determination of biotin levels in pharmaceutical
preparations, as w e l l as in f o o d and f o o d supplement products, w h i c h
constitute the main source of biotin in h u m a n s . For this reason, analytical
m e t h o d s h a v e b e e n developed, in order to determine biotin in biological fluids,
as w e l l as various kinds of food products and pharmaceutical preparation
c o n t a i n i n g biotin. M a n y m e t h o d s h a v e b e e n described for the determination of
1 1 1

2 - 5

MARCKI. DFKKUR, 1

2 7 0 M a d i s o n A v e n u e , N e w Y o r k , N c w Y o r k 1001ft

3
ORDER

Biotin D i s s o l u t i o n

513
l6

8i

biotin, using m i c r o b i o l o g i c a l ~ and, recently, high performance liquid

chromatographic ( H P L C ) p r o c e d u r e s . Although the microbiological
assays provide excellent sensitivity, these methods are laborious a n d t i m e
consuming, with an average incubation
time of 1824 nr. On the other hand, at present, the best H P L C procedures
involve electrochemical detection - ' and fluorescent reaction with derivatization.
Although these methods are highly sensitive and specific, they are
susceptible to interference, have long retention times, or use m o r e
sophisticated equipment, w h i c h is n o t normally found in a c o m m o n clinical
laboratory.
Drug dissolution testing is an integral part ' of pharmaceutical
development and in routine quality control monitoring of drug release
characteristics. To the best of our knowledge, no method has been reported
in the literature for the determination of BI release from pharmaceutical
dosage forms. Therefore, the objectives of this investigation w e r e to develop
a new, simple, fully automated, rapid, and sensitive procedure, which
involves no derivatization H P L C , with photodiode array detectors, for the
quantitation of BI in pharmaceutical formulation, tablets, and to evaluate its
in vitro dissolution rate.
1

9-141

9 1

t l 0 _ 1 3 ]

EXPERIMENTAL
Chemicals and Reagents
T h e H P L C grade acetonitrile, phosphoric acid 8 5 % , and hydrochloric
acid were purchased from Fisher Scientific Co. (Fairlawn, NJ, U S A ) . D i sodium hydrogen phosphate w a s supplied from Fluka (Buchs, Switzerland).
H P L C grade water w a s prepared by reverse osmosis and further purified by
passing through a Milli-Q System (Millipore Company, Milford, M A , U S A ) .
Pure BI w a s purchased from Sigma Chemical Co. (St. Louis, M O , U S A ) ,
B I O T I N tablets, containing 0.8 mg biotin per tablet, were obtained from the
local market. A stock solution of BI (2 | x g / m L ) was prepared in 0.02N disodium hydrogen phosphate ( N a H P 0 4 ) , p H 7 . 4 adjusted with phosphoric
acid. This stock solution w a s prepared weekly and further diluted to produce
concentrations of BI that ranged from 0.01 to 2.00 M-g/mL ( 0 . 0 1 , 0 . 0 5 , 0 . 1 , 0.2,
0.4, 0 . 6 , 0 . 8 , 1 . 6 , and 2.00 p , g / m L ) . T h e stock solutions were stored in the dark
at 70C until needed during the week. No instabilities were observed from
solutions stored under these conditions compared to fresh daily-diluted ones,
since there w a s no observed change in BI peak height or appearance of any
impurities.
(8,

2 7 0 M a d i s o n A v e n u e , N t w York, N e w York I fX) 10

ORDER

REPRINTS

514

A b o u l - E n e i n e t al.

Instrument and Chromatographic Conditions

A High-Performance Liquid Chromatographic System (HPLC), Waters
Alliance, dissolution system (Waters Associates, Inc. Milford, M A . U S A )
consisting of W a t e r s 2 6 9 0 D Separation Module with eight-needle dissolution
dispenser, Waters Transfer Module, with eight syringes, one dissolution test
bath (Hanson Research SR8-Plus), eight Uni-Probes, one for each dissolution
vessel to be sampled, and Waters 996 Photodiode array detector. T h e compound
of interest w a s detected at 190 n m . T h e data were collected with a M i l l e n n i u m
Chromatography M a n a g e r data collection system, utilizing a Pentium 4
computer connected to Inkjet HP PSC 750 printer. BI determination was
performed using a stainless steel Supelcosil LC-18 column (25 cm x 4.6 m m ;
5 p.m particle size) preceded by a Sentry guard column. T h e mobile phase
consisted of an 8 0 % aqueous solution (pH 2.S adjusted with phosphoric acid),
containing 2 0 % acetonirrile delivered at a flow rate of 1.6 m L / m i n .
32

In Vitro Dissolution Studies

T h e dissolution rates of BI from tablets were performed on a Hanson SR8Plus dissolution apparatus (Hanson Research Corp., Chatsworth, C A , U S A ) .
Drug release tests were carried out according to conventional U S P 26
dissolution procedures for the single-entity p r o d u c t s ,
with the use of a
paddle-stirrer type of apparatus, in 500 mL of 0.02N di-sodium hydrogen
phosphate ( N a H P 0 ) ( p H 7 . 4 using phosphoric acid, to simulate intestinal
medium), at a stirring rate of 75 r p m for 5 hr, then at infinity ( 2 5 0 r p m ) for the
rest of the dissolution period 5.5 hr. The temperature of the cell was maintained
at 37 + 0.5C by using a thermostatic bath. At each sample time interval, an
exact volume of sample was withdrawn from each flask and immediately
replaced with an identical volume of fresh medium to maintain a dissolution
sink condition. A correction factor was included in the calculations to account
for the drug lost in the samples. At predetermined time intervals (0, 0.083,
0 . 1 6 6 , 0 . 3 3 3 , 0.50, 0.75, 1, 1.5, 2 , 3 , 4 , 5 , and 5.5 hr), the concentrations of BI
(p,g/mL) in the dissolution medium were determined by the proposed H P L C .
1151

Analysis of Pharmaceutical Dosage Form

Preparation of Biotin Standard Solution

A stock solution of pure BI (2 u.g/mL) was prepared in water. This stock

solution was further diluted weekly with 0.02N di-sodium hydrogen

t!

M A R C E I . DEKKKR,

fNC

2 7 0 M a d i s o n A v e n u e , N e w York, N e w York 1 0 0 Wi

B i o t i n Dissolution

515

p h o s p h a t e ( N a H P 0 ) p H 7.4 adjusted with p h o s p h o r i c acid t o p r o d u c e

concentrations of BI that ranged from (0.01 to 2.00 n-g/mL) 0 . 0 1 , 0.05, 0 . 1 ,
0 . 2 , 0 . 4 , 0 . 6 , 0 . 8 , 1.6, a n d 2 . 0 0 M-g/mL.
2

Preparation of Biotin Solution from Tablets

T h r e e different s t o c k s o l u t i o n s o f B I O T I N t a b l e t s c o n t a i n i n g ( 0 . 8 m g )
biotin was prepared in 500 mL of 0.02N di-sodium hydrogen phosphate
(Na HP04) pH 7.4 adjusted w i t h p h o s p h o r i c acid to p r o d u c e concentration of
( 1 . 6 f A g / m L ) . F o r t h e c o n c e n t r a t i o n s 1.6 u , g / m L , w e f o u n d a v e r a g e a m o u n t s
p e r tablet (drug content) S.D. were 0.81 + 0.01 m g , while the percentage
a v e r a g e recovery + S.D. of BI tablets c o m p a r e d to the p u r e BI w e r e
1 0 1 . 2 5 1.25 (n = 3 ) .
2

Data Analysis
All data w e r e reported as the m e a n S.D. of at least seven parallel
studies. T h e results were calculated by linear regression without weighing,
u s i n g t h e f o r m u l a : Y = a 4- bX, W h e r e F i s t h e p e a k h e i g h t of t h e d r u g , a is t h e
intercept, b is the slope, and X is the concentration of BI. T h e a m o u n t of B I ,
obtained from the drug dissolution studies, was calculated from the calculated
l i n e a r r e g r e s s i o n e q u a t i o n . T h e i n v i t r o d i s s o l u t i o n d a t a ( n = 7 ) w e r e fitted t o
P e p p a s ' equation - to determine the kinetic order of release.
116

Moo
W h e r e M,/M<x, i s t h e f r a c t i o n a l d r u g r e l e a s e d , K i s t h e r e l e a s e k i n e t i c
c o n s t a n t , t i s t h e r e l e a s e t i m e , a n d n i s t h e r e l e a s e e x p o n e n t c h a r a c t e r i s t i c for
the drug. T h e release kinetic constant w a s calculated from the equation that
b e s t fit t h e r e l e a s e d a t a a n d , a c c o r d i n g l y , t h e t i m e for 5 0 % o f t h e d r u g t o b e
released was calculated ( T % ) . T h e student's Mest w a s used to determine
s t a t i s t i c a l l y significant d i f f e r e n c e s (p < 0 . 0 5 ) .
5 0

RESULTS AND DISCUSSION

A s p e c i f i c a n d r e p r o d u c i b l e fully a u t o m a t e d H P L C m e t h o d for t h e
analysis of biotin was developed. T h e method w a s validated under the
conditions described, BI retention t i m e w a s 3.90 0.2 m i n . T h e correlation
coefficient ( r ) w a s > 0 . 9 9 9 0 . 0 0 1 f o r t h e t e s t e d c o n c e n t r a t i o n r a n g e f r o m
0 . 0 1 t o 2 . 0 j j L g / m L ( n = 7 ) . T h e d e t e c t i o n l i m i t u n d e r t h e s e c o n d i t i o n s , defined

?
%
.,
'~
-J
SA

8>

2 7 0 Madison Avenue, N e w Y o r k . N e w York HXH6

ORDER

516

A b o u l - E n e i n e t al.

as t h r e e t i m e s the level of noise for-BI in b l a n k s a m p l e s , w a s 0.005 | i g / m L ,

w h i l e t h e quantification limit o f this m e t h o d w a s 0.01 | x g / m L . T o d e m o n s t r a t e
t h e utility of the m e t h o d , Fig. 1 d e p i c t s t h r e e representative c h r o m a t o g r a m s
i n c l u d i n g a blank s a m p l e (A); a s a m p l e s u p p l e m e n t e d (B) with 0.8 p , g / m L of
B I , and t h e third sample ( C ) , calculated c o n c e n t r a t i o n of 1.6 | x g / m L collected
1.5 hr after starting the tablet dissolution.

-0.005
0.00

2.00
4.00
Minutes

6.00

0.030

2.00
4.00
Minutes

6.00

Figure 1.
Chromatograms of a blank s a m p l e ( A ) ; a sample supplemented ( B ) w i t h
0.8 | x g / m L of pure BI powder; and the third s a m p l e (C), calculated concentration of
1.6 ( j L g / m L collected 1.5 hr after starting the tablet dissolution.

2 7 0 M a d i s o n A v e n u e , N e w York, N e w Y o r k tOOlfj

Biotin Dissolution

517

2.00 4.00
Minutes

Figure 1.

6.00

Continued..

t a b l e 1 shows the m e a n BI content in the tablets d o s a g e form. F o r

t h e t e s t e d t a b l e t s , n = 7 , (0.8 g m / t a b l e t ) , t h e m e a n d r u g c o n t e n t w a s
0.81 + 0 . 0 6 m g .
T h e dissolution study of B I O T I N * tablets revealed that BI in U S P m e d i a
( p H 1.2) s h o w e d n o d i s s o l u t i o n u p t o 3 hr, s i n c e B I i s v e r y s l i g h t l y s o l u b l e i n
w a t e r b u t it d i s s o l v e s in d i l u t e a l k a l i n e s o l u t i o n s . H o w e v e r , a first o r d e r r e l e a s e
k i n e t i c , r = 0 . 9 9 , w i t h d i s s o l u t i o n T s o of 14 1.3 m i n , w a s o b s e r v e d in U S P
media ( p H 7 . 4 ) as s h o w n in Fig. 2. For each plotted point, the m e a n reading of
eight tablets is s h o w n expressed as percent BI released.
T h e d i s s o l u t i o n d a t a o f B I , a t this p H , s h o w e d a little h i g h e r R S D % (9.3 t o
1 2 . 7 % ) a t l o w e r d r u g c o n c e n t r a t i o n s ( < 1.0 u - g / m L ) . O n t h e o t h e r h a n d ,
t h e R S D % w a s 3 . 6 - 7 . 2 % f o r BT c o n c B n t r a t i n n s > 1.0 [i.g/rnT. Thf.re.fnny
t h e utilized a u t o m a t e d d i s s o l u t i o n - H P L C s y s t e m p r o d u c e d p r e c i s e a n d

Table 1.
T h e m e a n * ( + S D ) biotin content in tablet
d o s a g e form.
L a b e l c l a i m ( m g p e r tablet*)
Measured amount (mg/tablet)
T h e % total a m o u n t per tablet
dosage form

'

0.8
0.81 ( 0 . 0 0 6 )
1 0 1 . 2 5 ( 0 . 7 2 )

13
S
%
.a>

'

M A H C T ; I . D B K . K E R . , YKC:.

27(3 M a d i s o n A v e n u e , N e w Y o r k . N e w Y o r k 1 0 0 1 6

Aboul-Enein et al.

518

nop

T i m e (min)
Figure 2.
T h e in vitro dissolution profile of B I O T I N * c a p s u l e s in U S P intestinal
m e d i a ( p H 7 . 4 ) . A v e r a g e of 7 determinations.

r e p r o d u c i b l e results. A l m o s t all t h e d r u g w a s r e l e a s e d (at p H 7 . 4 $ from t h e

tablets w i t h i n 1.5 hr.
I n c o n c l u s i o n , t h e fully a u t o m a t e d d i s s o l u t i o n - H P L C s y s t e m used w a s
rapid, r e p r o d u c i b l e , a n d c o n v e n i e n t t o follow B I after dissolution from
p h a r m a c e u t i c a l d o s a g e f o r m s , a n d required n o internal standard. T h e
m e t h o d d e s c r i b e d is suitable for quality control of biotin p h a r m a c e u t i c a l
formulations.

REFERENCES
1. L i v a n i o u , E.; C o s t o p o u l o u , D . ; Vassiliavou, L.; L e o n d i a d i s , L . ;
N y a l a l a , J . O . ; Ithakissios, U.S.; Evangelatos, J . P . A n a l y t i c a l t e c h n i q u e
for d e t e r m i n i n g biotin. J. C h r o m a t o g r . A 2 0 0 0 , 881, 3 3 1 - 3 4 3 .
2. M o c k , D . M . Biotin. In Handbook of Vitamins, 3rd E d . ; R u c h e r , R . B . ,
Suttie, J . W . , M c c o r m i l l e , D . B . , Maachlin, L J . , E d s . ; M a r c e l D e k k e r Inc.:
N e w York, 2001.
3 . B o n j o u r , J.P. B i o t i n i n h u m a n nutrition. A n n . N . Y . A c a d . Sci. 1 9 8 5 , 447,
97-104.
4 . W h i t e h e a d , C . C . A s s e s s m e n t o f biotin deficiency i n a n i m a l s . A n n . N . Y .
A c a d . S c i . 1 9 8 5 , 447, 8 6 - 9 6 .
5. W a t k i n s , B . A . Influences of biotin deficiency a n d dietary trans-fatty acids
on t i s s u e lipids in children. British J. Nutr. 1 9 8 9 , 61, 9 9 - 1 1 1 .

M A K C K I . DKKK&H, IN*;.

2 7 0 M a d i s o n Avenue, Ne-wYork. N e w Y o r k 1 0 0 1 6

<4

9
ORDER

Biotin Dissolution

519

6 . W a l l e r , J.R. I n c r e a s e d sensitivity o f t h e m i c r o b i o l o g i c a l a s s a y for b i o t i n

by Lactobacillus plantarum. A p p l . M i c r o b i o l . 1 9 7 0 , 20, 4 8 5 - 4 9 1 .
7. Voigt, M . N . ; W a r e , G.O.; Eitenmiller, R.R. C o m p u t e r p r o g r a m s for the
evaluation of vitamin B data obtained by microbiological methods.
J. Agric. Food Chem. 1978, 27, 1 3 0 5 - 1 3 1 1 .
8. I z u m i , Y.; Tani, Y.; Ogata, K. Microbiological biosynthesis of biotin.
M e t h . E n z y m o l . 1 9 7 9 , 62, 3 2 6 - 3 3 8 .
9 . K a m a t a , K.; H a g i w a r a , T . ; T a k a h a s h i , M . ; U e h a r a , S.; N a k a v a m a . K . :
Akiyama, K. Determination of biotin in multivitamin pharmaceutical
preparations by high-performance liquid chromatography with electroc h e m i c a l d e t e c t i o n . J . C h r o m a t o g r . 1 9 8 6 , 356, 3 2 6 - 3 3 0 .
10. H a y a k a w a , K.; O i z u m i , J . D e t e r m i n a t i o n o f f r e e b i o t i n i n p l a s m a b y l i q u i d
c h r o m a t o g r a p h y w i t h n u o r i m e t r i c d e t e c t i o n . J . C h r o m a t o g r . 1 9 8 7 , 413,
247-250.
1 1 . W o l f , J . H . ; Korf, J . 4 - B r o m o m e t h y l - 7 - m e t h o x y c o u m a r i n a n d a n a l o g u e s
as derivatization agents for high-performance liquid chromatography
d e t e r m i n a t i o n s : a r e v i e w . J. P h a r m . B i o m e d . A n a l . 1 9 9 2 , 10, 9 9 1 0 7 .
12. S t e i n , J.; H a h n , A . ; L e m b c k e , B . ; R e h n e r , G . H i g h - p e r f o r m a n c e liquid
c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f b i o t i n i n b i o l o g i c a l m a t e r i a l s after
c r o w n ether-catalyzed fluorescence derivatization with panacyl bromide.
A n a l . B i o c h e m . 1 9 9 2 , 200, 8 9 - 9 4 .
1 3 . N o j i r i , S.; K a m a t a , K.; N i s h i j i m a , M . F l u o r e s c e n c e d e t e c t i o n o f biotin
using post-column derivatization with O P A in h i g h performance liquid
c h r o m a t o g r a p h y . J . P h a r m . B i o m e d . A n a l . 1 9 9 8 , 16, 1 3 5 7 - 1 3 6 2 .
14. E k p e , A . E . ; H a z e n , C . L i q u i d c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f biotin i n
m u l t i v i t a m i n - m u l t i m i n e r a l tablets. J . P h a r m . B i o m e d . A n a l . 1 9 9 8 , 16,
1311-1315.
1 5 . United States Pharmacopeia (USP 26) and the National Formulary (NF
21); U n i t e d P h a r m a c o p e i a l C o n v e n t i o n I n c . : R o c k v i l l e , M D , U S A , 2 0 0 2 ;
2155-2156.
16. P e p p a s , N . A . A n a l y s i s o f F i c k i a n a n d n o n - F i c k i a n d r u g r e l e a s e from t h e
p o l y m e r s . P l i a i m . A c t a H e i v 19HS, 6 0 , 1 1 0 1 1 1 .

Received September 11, 2003

Accepted October 22, 2003
Manuscript 6227

3
a

MAROEI. DEKUIR,

INC.

2 7 0 M a d i s o n A v e n u e , N e w York, N e w York 1 0 0 1 6

Request Permission or Order Reprints Instantly!

Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article's rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly; Simply click on the "Request Permission/
Order Reprints" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers'
(AAP) website for guidelines on Fair Use in the Classroom.
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as

part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our Website
User Agreement for more details.

Request Permission/Order Reprints

Reprints of this article can also be ordered at
http://www.dekker.eom/servlet/product/DOI/l 01081JLC120027622