Quantitation of yohimbine utilizing capillary electrophoresis. Method development through statistical design.

Rochester Institute of Technology

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Theses

Thesis/Dissertation Collections

11-1-1999

Quantitation of yohimbine utilizing capillary

electrophoresis. Method development through

statistical design.

Jonathan Cooper

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Recommended Citation

Quantitation

of Yohimbine

Utilizing Capillary Electrophoresis.

Method Development through Statistical Design.

Jonathan W. Cooper

Novembe~,

1999

Thesis

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

APPROVED:

Paul A. Craig

Project Advisor

G. A.

Takacs

Department Head

Rochester Institute of Technology

Rochester, New York 14623

Copyright Release Fonn

QUANTITATION OF YOHIMBINE UTILIZING CAPILLARY ELECTROPHORESIS.

METHOD DEVELOPMENT THROUGH STATISTICAL DESIGN.

I, Jonathan William Cooper, hereby grant pennission to the Wallace Library of the

Rochester Institute of Technology to reproduce my thesis in whole or in part. Any

reproduction will not be for commercial use or profit.

Date:

Iz/1/9~

I

ACKNOWLEDGEMENTS.

Iwould

like

to thank _myadvisor, Dr. Paul

Craig,

withoutwhomthis researchwould not

have been

possible. _{His continuing} support and suggestion were essential towards

driving

this project _{to completion,} and

his faith in

_my abilities allowed me to begin to

discoverand

develop

_myownprofessional

interests.

Many

thanks toDr. Carol Marchetti

for

her

contributions to the statistical aspects of this study. Her enthusiasm and

motivation

helped

to turn a _seemingly

imposing

statistical tool into an

interesting

and

beneficial

challenge, onethatIhopeothers willfollow my footstepstoward. Iwould also

like

to extend _my gratitude to James

Wesley

for

helping

direct the path of this

investigation,

and Dr. T. C. Morrill for his guidance

during

_my graduate career. I thank

the Rochester Institute of

Technology

Department of

Chemistry

for

_giving me the

opportunity to earn this

degree,

as well as _providing me with a solid educational

foundation fromwhichIcanexpand.

I thank_my

family

for

theirunconditional love and understanding. These past

few

years

have

_{been challenging}

for

all of us

in

ways other_{than academic,}

but

somehow we

have

been able to maintain and even

develop

our unique relationships. As I complete this

project and

look

toward the

future,

there are a

large

number of uncertainties that I will

ACKNOWLEDGEMENTS.

I would

like

to thank_{my advisor, Dr. Paul}

Craig,

without whomthis research would not

have been

possible. _{His continuing} support and suggestion were essential towards

driving

this project _{to completion,} and

his faith

_{in my} abilities allowed me to begin to

discover

and

develop

_myown professionalinterests.

Many

thankstoDr. Carol Marchetti

for

her contributions to the statistical aspects of this study. Her enthusiasm and

motivation

helped

to turn a _seemingly

imposing

statistical tool

into

an

interesting

and

beneficialchallenge,onethatI hopeotherswillfollow my

footsteps

toward. Iwould also

like

to extend _my gratitude to James

Wesley

for

helping

direct the path of this

investigation,

andDr. T. C. Morrill for his guidance

during

_my graduate career. I thank

the Rochester Institute of

Technology

Department of

Chemistry

for giving me the

opportunity to earn this

degree,

as well as _providing me with a solid educational

foundation fromwhichI canexpand.

Ithank _my

family

fortheir unconditional love and understanding. These pastfew years

have

_{been challenging}

for

all of us inways other_{than academic,}

but

somehow we

have

been able to maintain and even

develop

our unique relationships. As I complete this

project and

look

towardthe

future,

there are a large number of uncertainties that I will

TABLE

OF CONTENTS.

ABSTRACT

i

INTRODUCTION

1

Yohimbine

Background 1

History

of

Capillary

Electrophoresis 4

Theory

of

Capillary

Electrophoresis

7

Modesof

Capillary

Electrophoresis 10

Factorial Experimental Design 13

Summary

19

Definitions 20

MATERIALS AND METHODS 23

Reagents 23

Instrumentation 24

Commercially

AvailableProducts 25

Methods 27

ExperimentalDesign

27

PreparationofBuffers

for

Capillary

Electrophoresis

32

Methodsof

Extraction

35

Optimized Extraction

of

Commercial Samples

40

Instrumental Methods

41

RESULTS 44

Factorial Design

One

(Fractional)

44

Factorial Design Two

(Full)

50

Optimized Experimental Conditions 54

Commercially

Available Products 55

DISCUSSION 59

Factorial Design One

(Fractional)

59

FactorialDesign Two

(Full)

69

Optimized Experimental Conditions 71

Commercially

Available Products 72

Summary

72

Future Work 73

REFERENCES 74

FIGURES.

Figure

1.

Molecular

structure of yohimbine 2

Figure 2. CE Schematic 7

Figure 3.

Capillary

wall

interactions

9

Figure 4. Reverse

laminar

flow

diagram

10

Figure 5. 'Plug-like'

flow diagram

10

Figure 6. Designgeneratorsfor factorial designone 28

Figure 7. Run 13andRun 16

factorial design

one 46

Figure 8. Run 2andRun 14factorial designone 47

Figure 9. Maineffects plot

Area(2)

48

Figure 1 0. Run 1 5 andRun 1 6 factorial designone

49

Figure 1 1. Run 2 andRun 34

factorial design

one 50

Figure 12. Run 5 andRun 7 factorialdesigntwo 52

Figure 13. Run 5 andRun 6 factorial designtwo 53

Figure 14. Run 1 andRun 5 factorial designtwo 54

Figure 15. Yohimbinecalibrationcurve 56

TABLES.

Table 1. 23

factorialexperimental design 14

Table 2. 25 factorialexperimental

design

₁₆

Table3. y4 fractionof

25

Table 4. Factor levels

chosen

for factorial design

one 28

Table 5. Designmatrix

for factorial

design one 29

Table 6. Factor

levels

chosen

for factorial design

two 31

Table 7.

Design

matrix

for factorial design

two 31

Table 8. Methods

for factorial

designone 42

Table

9.

Methods for factorial

designtwo 42

Table 10. Method forcommercial analysis 43

Table 1 1. Descriptive statistics

for

factorial designone 44

Table 12. pvalues of

factorial

designone 45

Table 13. Descriptivestatisticsfor factorial designtwo 51

Table 1 4. pvaluesoffactorial designtwo 51

Table 15.

Commercially

availableproducts 55

Table 16. Tenextraction conditions 56

ABSTRACT.

In an effort to combat the _rising cost of

health

care

in

the United States

by

encouraging preventative and _{self-care, the}

federal

government passed the

Dietary

Supplement

Health

Education

Act of1994. This act _essentially

deregulated

the natural

product market _allowing

herbs,

including

_{yohimbine, to}

be

sold overthe counter without

undergoing the same strict evaluation as a traditional synthetic

drug

candidate. As the

full

potential ofthesenaturalproducts

is

realized,

including

possibleharmfulside _effects,

a need arises

for

the

identification

and quantitation of the active components from

commercial preparations.

Commercial samples were _{qualitatively} and _{quantitatively} analyzed for

yohimbine _utilizing a _capillary electrophoretic method optimized through statistical

design.

Calibration curves produced with this method _routinely achieved

R2

> 0.998.

Method reproducibility (n =

10)

and extraction _{efficiency (99.8% recovery)} were

determinedtobe sufficient. Itwas shownthata statistical designcould

limit

the amount

of experimental runs while _providing a significant amount of

information

about

INTRODUCTION.

Yohimbine Background.

The West African tree

Yohimbe is located

in the

low

_altitude,

jungle

forests

enveloping southwestern Nigeria.

Growing

from 20 to 50

feet

high,

this tall evergreen

forest

tree

has

_glabrous,

leathery

leaves

three to

five inches in

length with _upcurving

lateral nervesthat

fade

out at the margins. White

flowers,

containing winged _seeds, are

arranged

in

umbel-like clusters attheends oftheshoots

(1,

2). It

is

thecompounds

found

inthe

bark

ofthis tree that

have

historically

drawnattentiontowards theYohimbe.

Documentation of the

Bantu-speaking

people

describes

yohimbe as an

aphrodisiac andstimulant

traditionally

used

during

_mating ritualsthat _{last up}to 15 days.

There are several methods of_preparing yohimbe bark for use (2). A tea

is

prepared

by

boiling

yohimbe bark shavings in waterfor

less

than fourminutes. Heat

is

then turned

down,

allowing the brew to simmer

for

an additional 20 minutes. The shavings are

strained,andtheteaissipped onehour beforethedesiredeffects aretoberealized.

A method that leads to the effects in a shorter time after

ingestion involves

extractionofthebarkpowder witha

drinkable

alcohol. Aftera period ofeight

hours,

the

alcohol

is

strained and evaporated,

leaving

a residue that can

be

snuffed or taken

subcutaneously. Effects through this method are more _{pronounced, occurring} within

twenty

minutes (2).

The bark is reported to contain _up to 6% yohimbine

(Figure

1),

_along with a

number of similar indole alkaloids

including

_yohimbenine,

isoyohimbine

H,COOC

QH

[image:14.537.190.348.35.147.2]

Yohimbine

Figure 1. Molecularstructure ofYohimbine.

the

form

of the

hydrochloride is

also known as quebrachine,

isolated

from the South

American evergreenQuebracho tree

(2, 3)

as well as

from

Rauwolfia viridis

leaves

(4),

Rauwolfia caffra root bark and seeds

(5),

Rauwolfia serpentina root

(6),

Rauwolfia

oreogiton root

(7),

Rauwolfia volkensii root

(7),

Rauwolfia vomitoria root

(8),

and

Rauwolfiayunnanensis(9).

Yohimbine is the most

interesting

alkaloid

from

_{these sources,}

having

gained

notoriety in a number of circles as an alternative medicine treatment. Suggested for

humanuse in

bodybuilding

as an alternative to anabolic steroids

(1, 10),

yohimbine

has

been employed in _veterinary medicine as an antagonist for anesthesia and sedation of

animals, as well as

for

the treatment of impotent stallions

(11,

12). Classified as an

alpha-2-adrenergic

blocker,

this sympathomimetic

indole-type

alkaloid also

displays

serotonin

inhibiting

properties(1,2). In

both

humansand _animals,yohimbine produces a

complex patternofresponsesthat includeanti-diuresis and central _{excitation, comprising}

elevation ofblood pressure and

heart

rate, increased motor _activity and

irritability

(13).

Recent focus

has

beenconcerned withtheaphrodisiacproperties and use of yohimbine as

The _marketing of yohimbine

falls

under the

Dietary

Supplemental Health

Education

Act

(DSHEA)

of

1994,

a governmental effortto

limit

the _skyrocketing cost of

United

States health

care that

has

_essentially

deregulated

the natural product market.

Natural

products are not _strictly evaluated pre-market as a synthesized

drug

candidate

would

be,

and tend to

draw

_{scrutiny only} when adverse effects are noted as a result of

consumer use.

Regulatory

action toward another natural product with central nervous

system stimulant _{properties, ephedrine,} arose _only after toxic

levels

were witnessed

clinically

(36,

37). These complications _{may be due} to the inherent properties of the

sources

from

which these natural products are

harvested,

_namely vegetation. Alkaloid

quantitiesfromtwo

distinct

plants_{may be different from different}regions,quantities_may

be different betweentwodistinctplantsinthesameregion, orquantities_may

be

different

between

two leavesonthesametree. Apreparation of naturalproducts_may

in

turnhave

varying concentrations between

batches,

between

bottles,

and even between pills in the

same

bottle for

this reason. Thisis animportantconsiderationforacompoundthat

has

a

small range between a therapeutic dose and toxic

dose,

when product variance could

meanthedifference between lifeanddeath.

Asthe drawbacksoftheDSHEAare_eventuallyrealized and attention

focuses

on

dose

reproducibility, qualitative and quantitative characterization of _many natural

products,

including

yohimbine,will gainimportance. Efforts have thus

far been

reported

toward the identification and quantification of yohimbine

in

commercial products and

biological fluids _utilizing fluorescence

(6),

ultra-violet_spectroscopy

(UV) (5-8),

infrared

spectroscopy

(IR) (7, 8),

nuclear magneticresonance spectroscopy

(NMR)

(8),

thin-layer

mass _spectroscopy

(GC/MS)

(8, 13),

gas-liquid _{chromatography}

(GLC)

(5),

liquid

chromatography

(LC)

(40),

and

high-performance

liquid

_{chromatography}

(HPLC)

(6, 13,

39-44). There

have been

no reported

investigations

of yohimbine _{utilizing capillary}

electrophoresis, a technique that

is

_{swiftly gaining} prominence in the analytical

laboratory.

History

of

Capillary

Electrophoresis.

Inthe 1930's Arne Tiselius developed the _{first moving}

boundary

electrophoresis

method_utilizinganopenU-shapedquartzcolumn. Proteinmixtures in freesolution were

partially resolved as _{abutting bands detected}

by

ultraviolet absorbance (45).

However,

this _pioneering technique had

drawbacks

that included large sample _volumes, and an

incomplete separationoftheproteins.

Investigation ofimproved anticonvective media in subsequent

decades directed

the

focus

toward zone electrophoresis. Polyacrylamide _gels,

developed in

the

late

1960's,

contain _stacking and _{resolving buffer} systems that yield native protein and

sodium dodecyl sulfate

(SDS)

boundprotein separations (46). Knownas polyacrylamide

gel electrophoresis

(PAGE)

and

SDS-PAGE,

these techniques continue to give

high-resolution separationsasimportanttoolsin biochemical characterization.

Around the time PAGE was designed to separate proteins on the

basis

of charge

or _size, an investigation was _underway to

develop

a separation

based

on protein

isoelectric points. Isoelectric

focusing (IEF)

employs a gel _containing an

immobilized

deprotonated

(50). Hydrated

buffer

cations coordinateto the anions atthe _capillary wall

to

form

the rigid

Helmholtz

layer,

and the mobile Gouey-Chapman

layer

to complete

electroneutrality

(Figure

3). A

drop

in

negative _potential, described

by

the zetapotential,

characterizes the Gouey-Chapman

layer:

the magnitude of wall negative charge is

directly

proportionalto

layer

thickness. Whilethisregion extends_onlyafewnanometers

beyond

the Helmholtz

layer,

it

is

the mobile cations contained within that generatebulk

electroosmotic

flow.

Mobile hydratedcationsoutnumbermobileanionsin_{this area,}

Helmholtz Layer

|

Gouey-Chapman

Outer-Helmholtz

Region

Figure3. Interactionsatthe_capillarywallthatfuelelectroosmotic

flow. Notshown:hydratedanionsinthediffuse layer (Outer-Helmholtzregion).

resulting in a net

flow

toward the cathode under an applied voltage.

Immediately

upon

the application of _voltage, cations in this layer will have a greater _velocity then the

remainderof_{the capillary} cross section. The resultantflow profile _may

have

a parabolic

character asthe electrophoretic _mobility ofcations inthe

diffuse

portion ofthe _capillary

are _partially shielded

by

coordinated mobile anions with an opposite pe.

This

phenomenon would appear similartoalaminar

flow

profiletowards the anode,

but in fact

[image:17.537.105.415.262.405.2]

[image:18.537.112.422.93.167.2]

'plug-}

Figure 4. Reverse laminar flowdiagram

like'

flow is

obtained at _any cross sectional area

beyond

the Helmholtz plane, as this

pulling force

is

uniformforthelengthofthecapillary. Cation

flow

nearthe_capillarywall

inside

the rigid Helmholtz layer approaches _{zero, resulting in} the accepted depiction of

electrically driven flow (Figure 5).

r

]

p.

+|

>

h

T_

, ,

:- *

Figure 5.'Plug-like'electroosmoticflow

Modesof

Capillary

Electrophoresis.

The term capillary electrophoresis describes the

family

of separation techniques

based

on_{the mobility} differences of charged species under an applied voltage.

Mention

shouldbe givento the variations onthis empirical analytical technique that

have

gained [image:18.537.113.424.402.476.2]

popularity toward specialized analysis. _{A summary} of the notable techniques and

applications

follows.

Capillary

Zone

Electrophoresis

(CZE).

Capillary

zone electrophoresis

is

theelectrophoretic separation of

ions

ina

buffer

solution under an applied voltage.

Micellar Kinetic

Capillary Chromatography

(MEKC).

Upon the addition of anionic surfactant molecules above a critical micelle

concentration

(CMC),

hydrophobic

tails ofthese molecules _{coordinate, exposing} their

hydrophilic heads and _assuming a spherical shape characteristic of a micelle. These

micelles

have

a ue toward the anode. Analytes inthiselectrophoretic system are further

resolved on the basis of

hydrophobicity

as

interaction

with the micelles

delays

the

observedmigration.

Capillary

Gel Electrophoresis (CGE).

The capillary

is

loadedwith a_gel, such as _{polyacrylamide, commonly} employed

in

PAGE. Analytes are then separated based on charge to size ratio under an applied

voltage. This technique

has

a high resolving power

for

_{proteins, peptides,} and amino

acids.

Capillary

Isoelectric

Focusing

(CIEF).

An electrolyte

containing

a mixture of ampholytes and sample

is

loaded onto the

column.

When

voltage

is

_applied, the ampholytes migrate to form a continuous pH

gradient

in

thecapillary.

Analytes

migrateto thelocationon_capillarythatcorrespondsto

their pi. Thevoltageis_removed, and _capillarycontentsare elutedpastthedetectorunder

an applied pressure.

Capillary

Isotachophoresis (CITP).

This

is

a _moving

boundary

electrophoretic technique. The sample _plug

is

introduced after a

leading

electrolyte with _{mobility faster} than the fastest analyte, and

before a

terminating

electrolyte of slower_mobility than the slowest analyte. Compounds

are resolved withinthe sample_plugasitmigratestowardthedetectionwindow.

Capillary

Electrochromatography

(CEC).

CEC is a_capillaryelectrophoretic technique_utilizing a_capillary packed with one

of_anynumber of_stationaryphases used in HPLC. This technique can _{employpressure,}

voltage, oracombinationofthe two asthe

driving

separationforce. This isuseful inthe

separation ofcharged analytes that differ

in hydrophobicity.

The

investigation

ofthis

technique has aided

in

the advancement ofmicro-HPLC _systems, as _pump

technology

advances.

Factorial Experimental Design.

Recently,

statistical experimental

designs have been

increasingly

employed as a

useful option

in

analytical method

development.

A factorial design

is

equipped to

address several major experimental concerns

for

which the traditional One Factor at a

Time

(OFAAT)

trialand error

development

technique

is

not suited(52).

Jointeffects or

interactions

aretheeffectthat the simultaneous variationoftwo or

more experimental parameters will

have

on response. These effects can _{only be}

identified and studied

by

the correlative change ofthe selected

factors.

The OFAAT

method

involves

_{systematically optimizing} one experimental _variable, while

holding

all

other variables constant. The method is then optimized for a second _variable, again

keeping

all other variables constant. Subsequent variables are investigated in the same

manner. To evaluatejointeffects withthis

design,

_{it is necessary} to reevaluate variable

oneupon optimizationofvariable

two,

then thereevaluation of variabletwowithchanges

in variable one, eventually moving through all variables

in

this systematic approach.

Clearly,

the number of experimental runs would _quickly

become

_exhausting as the

number ofinvestigatedvariablesincreased.

A factorial experimental design allows for the systematic

investigation

of

joint

effects

by

_enablingastatisticalanalysistointerpretthesignificanceof

factor

levels,

while

limiting

the number ofexperimental runs needed. This

design

is useful when _expense,

quantityofmaterialortimeare

limited,

as experimentnumbers are_{significantly}reduced.

There are several software programs that are capable of

handling

the complex

calculations to analyze data from a factorial

design,

but it is useful to understand

how

thesevaluesarecomputedto_employthis technique.

development

ofthe method

for synthesizing

ampholyte mixtures

for

high-resolution IEF

in

1969,

still a separationtechnique

found

inthemodern

biochemistry laboratory

(47).

O'Farrell

combinedIEFwith

SDS-PAGE in

the

1970's,

_creatingtwo-dimensional

electrophoresis, a technique capable of _resolving more than 50 components in each

direction.

Utilizing

an

isoelectric

point separation followed

by

an electrophoretic

separation

in

the perpendicular

direction,

several thousand compounds can

be

isolated

withthis approach(48).

During

the

late

1970's,

the

family

of gel electrophoretic techniques gained

acceptancein

biochemistry

laboratories,

_opening

doors

for investigationofprotein-based

biopharmaceuticals.

Meanwhile,

high performance

liquid

_{chromatography}

(HPLC)

was

gaining notoriety as an analytical tool in pharmaceutical and chemical laboratories.

Along

withhigh_resolution, HPLC offeredease of_automation,andexcellent quantitative

precision for the determination of low molecular weight compounds and

industrial

polymers (49). As gel techniques were applied in an industrial _{setting, their} detection

limitations and inherent

lengthy,

involved

preparation procedures

did

not compare

favorably

to the swift and reliable chromatographic techniques employed at the time.

This prompted pharmaceutical and bioanalytical chemists in these

fields

to turn their

attentionto HPLC for biomolecule characterization as _{protein, peptide,} and nucleic acid

chromatographic mediabecame available

during

the_early

1980's,

(50).

A strong environmental awareness movement occurred in the

1980's

as the

biotechnology

field broadened. This

involved

a chain of events _effectively

increasing

waste disposalcosts. Amain analyticaltoolatthis

time,

HPLC,

_routinelygenerates

large

Consider

the case of a

full

factorial

2k

= 23

design:

variation ofthree

factors (k

=

3)

at two

levels.

_{For example;} the effect oftemperature (factor A:

25,

37),

pressure

(factor B: 1 _atm, 3 _atm), and wavelength(factor C: 300 nm, 500 nm) on photosynthesis

by

a _newly

discovered

algae. An experimental run chart is assembled and completed, [image:23.537.55.470.228.359.2]

recording theresponse (Table 1). Therearefoursets ofexperimentalruns where factor

Table 1. 23

Factorialexperimentaldesign. Threefactors

(A,B,C)

attwolevels: low

(-)

andhigh (+).

Run Number Factor A Factor B FactorC Response

(X)

1 - -

-X,

2 + -

-x2

3 - +

-x3

4 + +

-x4

5 - - ₊

x5

6 + - +

x6

7 - _{+ +}

x7

8 + + +

x8

A

is

varied as

factors

B andC remained constant. Runs 1 and

2,

where Achanges while

B andC areboth negative:Runs 3 and

4,

where B

is

positive, and C isnegative: Runs 5

and

6,

where B is negative and C is positive: Runs 7 and

8,

where B and C are both

positive.

Therefore,

theeffect offactor Acanbe describedthroughEquation 2.

Effect A=

((X2-XQ

+

(X4-X3)

+

(X6-X5)

+

(Xg-X?))

4

Thenumber generateddescribestheeffectof_{moving from}a

low

toa

high level

in

A,

regardlessofthe levels ofB andC. Thiscalculationcan

be

completed

for

each ofthe

factors

employedinafactorial designtodeterminetheireffect

(52).

Joint

effects are

handled in

a similar manner. Forthis analysis, the

design has

two

pairs of

data

points with a variation of

A;

one atthe

high

level of

B,

the other at the low

level,

while C remains constant. Halfofthe

difference

ofthese two pairs isthevalue of

theAB

interaction (Equation

3).

Effect Aat positiveB=

((X

-X7)

+

(Xfi

-Xs))

2

Effect AatnegativeB=

((X*

-X,)

+

(X,

-X^)

2

Effect AB=_{((Effect A}

atpositive

B)

-(Effect Aat negative

B))

3

2

Scheme 1.

This calculation can be completed for each factor of interest to

determine

the

magnitude of joint effects.

Utilizing

computer software in the

handling

of these

equations

dramatically

lowersanalysistime

for

thisas well asa

larger

model.

Dueto the number offactorsthataffect a separation in

Capillary

Electrophoresis,

a factorial design presents an

interesting

choice

for

a

thoroughly

systematic method

development. Separation voltage,

temperature,

buffer

type,

buffer pH,

buffer ionic

strength, column

length,

organic modifier _{concentration,}

injection

type,

and

injection

setpoints are nine relevant factors. To investigate the main and

interaction

effects of

these ninefactorswitha

full

factorial

design,

k=

9,

and

29

or 512 runs would

be

needed.

It

is

possibletolowerthis number, utilizingafractional

factorial design

(55,

56).

Fractional

Factorial Design.

There are several advantages to _utilizing a fractional factorial design when the

numbers of experimental variables

in

a

factorial

investigation are large. There is

little

loss

of

information

while

significantly

lowering

the length of experimentation

(52,

53).

Considerthe

design

of a

2s

[image:25.537.72.469.223.690.2]

factorial

experiment(Table 2). This investigationwould

Table 2. 2 Factorialdesign.(Fivefactorsattwo_levels,centerpoints excluded).

Run Factor A Factor B Factor C Factor D FactorE Response

(X)

1 - - -

-^

2 + - - -

-x2

3 - ₊ - -

-x3

4 + + - -

-X4

5 - - + -

-x5

6 + - + -

-Xe

7 - + ₊ -

-x7

8 + + + -

-X8

9 - - - ₊

-x9

10 + - - +

-X10

11 - ₊ - ₊

-X11

12 + + - +

-X12

13 - - _{+ +}

-X13

14 + - + +

-X14

15 - + + +

-X15

16 + + + +

-X16

17 - - - - +

X17

18 + - - - ₊

X-I8

19 - + - - ₊

X19

20 + + - - ₊

X20

21 - - + - ₊

X21

22 + - ₊ - +

X22

23 - + + - ₊

X23

24 + + + - ₊

X24

25 - - - + +

X25

26 + - - + +

X26

27 - + - + +

X27

28 + + - + ₊

X28

29 - - + + +

X29

30 + - + + +

X30

31 - + + + +

X31

32 + + + + +

X32

requirethe collection of

32 data

points.

Generating

a

fractional

factorial

design

of a

%

fraction

ofthis tablewould require_only

8

experimental runs(Scheme

2),

witha

2(k-s)=2(5-2)=23 =

8

Where

k

-number of

factors

and s

is

derived fromtheequation

1/4=(l/2)s

Wherethe fraction=1/4.

Scheme 2. Generationof a%fractional factorial designoffive factorsattwolevels(k=

5,s= 2).

minimum ofinformation

loss

duetoconfounding.

Any

3

(k-s)

factors

are set_{up in}a

full

factorial

experiment. It is

first

_necessary to determine design generators (53). Design

generators represent the products of_multiplying together a combination ofthe + and

values from these first three columns. It _{is necessary} to utilize s number of unique

generators

for

a fractional factorial

design;

these generators must not be theproducts of

anyother generator combination(Scheme 3). Thevalues

for

the

fourth factor

column are

produced

D =

AC,

E=_AB

AC * AB=AACB=

CB

Scheme3. Designgeneratorsforfactors DandE. Theimplicit designgeneratoris_CB,theunique product ofDE.

by

multiplying the + or - in_columns A _and

C,

corresponding to the

design

generator

for

variable D and _{resulting in} a + or . The values

for

the

fifth factor

column are then

produced

by

multiplying

the + or

-in

columns A and

B,

_{corresponding} to

design

generatorE. Theresult

is

a V*

fraction

of a

25

[image:27.537.72.466.147.280.2]

factorial design

(Table 3).

Table 3. Vafractionof a

2s

factorial design.Generators D=

AC,E=_AB.

Run Factor A Factor B Factor C Factor D FactorE Response

(X)

1 - - - _{+ +}

Xi

2 + - - -

-x2

3 - + - ₊

-x3

4 + + - - +

x4

5 - - _{+ +}

-x5

6 + - _{+ +}

-x6

7 - _{+ +} - +

x7

8 + + + + +

x8

Therearethree additional quartersthatcanberepresented

by

thedesigngenerators D =

-AC andE=

-AB,D =AC and E=-AB, orD

=

-AC andE =AB. The quarter chosento

be

utilizedisarbitrary.

It

is

importanttounderstandandaccountfor confounding

factors.

Ifseveral

measurementsaremade whentwofactorsare atthelower

level,

and alsowhenthe two

factors

areat ahigh

level,

butnot whenthe

factors

are at

different

levels,

the effectdueto

the individual

factors

cannotbeestimated.

Any

information lost ina

fractional design

canbeattributed_{to these confounding} factors.

Resolutionof afractional factorial

design is determined

as the minimum number

of variables employedinthe design generators, a number thatrelates to the

confounding

offactor interactions. Intheexampleabovefora%

fraction

of a

25

design,

theresolution

(R)

is

calculated as R= _2. Effects

having

an order

less

than 2 are not confounded with

any othereffect with order

less

thant =_R

of

4,

main effects

(e

=

1)

are not confounded with _any othermain effect (t=

1)

or

two-factor

interaction (t = _2).

Two-factor interactions (e

=

2)

are not confounded with main

effects

but

_may

be

confounded with othertwo

factor interactions

(53).

When _approaching a complex analytical technique such as _capillary

electrophoresis,

it

may be

beneficial

toutilize afractional factorial designtowardmethod

development,

ruling out a large number ofinfluential factors with a limited amount of

experiments.

Summary.

At this

time,

there have been no reported studies _concerning the usefulness of

capillary electrophoresis in the qualitative and quantitative analysis ofyohimbine from

either_commerciallyavailable productsorbiologicalfluids. This paper willdeal withthe

investigation of _capillary electrophoresis as an analytical tool for the qualitative and

quantitative analysis of yohimbine in commercially available products. In _{addition, the}

use of a factorial experimental design will be evaluated to

investigate

nine important

factorsthatinfluence_capillary electrophoresis methoddevelopment.

DEFINITIONS IN CAPILLARY

ELECTROPHORESIS.

Area.

Thearea under a peak on an output electropherogram.

Capillary

Effective Length (I).

Lengthofthe_capillary employedfrom inletto detectorwindow.

Capillary

TotalLength (L).

Lengthofthe_capillaryemployedfrom inlettooutlet.

Electrophoretic

Mobility

(pe).

pe =

q/(6Tcr)r)

Where: _q=_ion

charge

r\ =

solution_viscosity

r=_ionic radius

Electrophoretic

Velocity

(vep).

Thisvalue

is

calculated

by dividing

theeffective _{capillary length}

by

themigration

time

Joule Heating.

Theresultant

heating

of aconductive mediumupon application of a current

flow.

Field Strength (E).

V L

Where:

V=

applied voltage

L =

total_capillary

length

Migration Time.

Migrationtime

is described

as the amount oftime

for

a compound to travel

from

the point of

injection

to the point ofdetection in capillary electrophoresis. This value is

analogous to a retention time _{in liquid chromatography (LC).}

However,

separations of

compounds in CE are a

function

of individual compound mobilities under an applied

voltage, where LC separations are afunction ofindividual compound

interactions

with a

stationaryphase.

Resolution(Rs).

Resolution is described as the separationbetween _{two peaks,} andrepresented

by

the equation:

Rs

=0.18Ape(EI)1/2

Pern

Symmetry.

Symmetry

is

ameasureof peak shape

deviation

from

a

Gaussian

curve.

Theoretical Plates (N).

Theoreticalplatesarethechromatographicdeterminationofseparation efficiency.

N= iteEI

2D

Where:pe =

Electrophoretic mobility D=_Diffusion

coefficient ofthesolute inthebuffersystem

E =_{Electric Field}

I =_Column

effectivelength

Zeta Potential (Q.

The zeta potential is a measurement ofthe potential difference between

the_capillarywallandthebuffersolution within.

thiswaste removal.

Renewed

interest

surfaced atthis time

for

thedevelopmentof

micro-bore

column

chromatography in

an effort to reduce solvent waste. The development of

this technique

theoretically

lowers

experimental costs while _maintainingresolution (50).

A combination of the positive aspects ofthese techniques are employed in _capillary

electrophoresis, which utilizes a charge

based

separation

in

a microbore column.

Hjerten

described

the

first

_capillary electrophoresis design in 1967 while at the

University

ofUppsala

in

Sweden (51).

Utilizing

1- to 3-mm internal diameter quartz

capillaries coated with _{methylcellulose,} separations ofinorganic

ions,

proteins, nucleic

acids, and microorganisms were demonstrated

by

free

zone electrophoresis and

later,

isoelectric

focusing.

Capillary

technology

appearedto be detrimentalto the evolution of

this

technique,

as variations in optical _pathway and

large

column

diameters

were the

limiting

factors

of performance (50). Gel techniques remained superior for these

applicationsduetothe_availabilityofthe_necessary

technology

atthe time.

During

the latterpart ofthe

1980's,

theinvestigation of_capillary electrophoresis

advanced

due

to the useful advantages ofsmall amount of solvent employed and the

qualitative precision obtained

in

analysis of

biopharmaceuticals.

This evolution was

aided

by

the

technology

found in gas chromatography, in the form of small

internal

diameter fused silica_capillaries, andfrom advancesin

HPLC,

_namely

improved detector

and autosampler capabilities. CE has since emerged as a technique _{complimentary} to

HPLC that offers _reliable, efficient separations

for

several unique applications

in

the

biotechnology

and biopharmaceutical

fields.

The separation of structural proteins

(49,

50),

restriction

digests

(49, 50),

andPCR products

(50)

are _only a

few

ofthe examples

Theory

of

Capillary

Electrophoresis.

Capillary

electrophoresis

(CE)

is

the

family

ofseparationtechniques

based

onthe

mobility

differences

of charged species in

free

solution under an applied voltage.

Typically,

a

fused

silica _capillary with an internal diameter between 25 and 100 um is

placed

between

two

buffer

reservoirs. _{The capillary is filled} with

buffer,

then a high

voltage

is

applied to the electrodes in each buffer _{reservoir, creating} an electric

field

across the length of the _{capillary (Figure 2).} It is within this electric field that a

separation

based

on charge to size ratios

is

afforded. Detection of resolved analytes is

achieved on-column througha window on the _{capillary from} whichthe external _coating

has been

removed.

v;

[image:33.537.117.403.374.534.2]

Electrophoretic Mobility.

Electrophoretic

_mobility

(pe)

is described

asthe_velocity that an

ion

demonstrates

under an applied voltage.

Magnitude

of_mobility

is

a

function

ofseparation buffer pH

effect on analyte charge.

Theoretically,

anions have _{pe towards the} anode, cations

have

Pe toward the cathode, and neutral analytes will

demonstrate

no pe-

However,

in the

capillary electrophoretic separation of a mixture of _{cations, anions,} and _neutrals, at a

neutral or alkaline _pH,peaks characteristic foreach species will

be

seen on theresultant

electropherogram. This result

infers

that regardless of_charge, each ion will have a net

mobility toward the cathode. Thephenomenon that contributes to thispeculiar effect is

called electroosmoticflow (EOF).

GenerationofElectroosmotic Flow.

Electroosmoticflow

(electroosmosis)

(Equation

1)

is

described

as abulk

flow

of

= sE

Atu\

Where: s=dielectric _{constant ofthe}

buffer

r| =

viscosityofthe

buffer

,

=_{zeta potential measuredatthe}

capillarywall

buffer solution under an applied voltage. This

flow is directed

toward the cathode in a

bare fused

silica_capillarywhenbufferpH

is

abovethree.

As buffer pH

is

increased betweenthree and _{eight, the} walls ofthe

fused

silica

capillary are _{proportionally}

deprotonated,

inferring

a negative charge on oxygen at the

MATERIALS AND

METHODS.

Reagents.

Standards.

Standard

Company

CAS# EEC#

Yohimbine hydrochloride Sigma 65-19-0 200-600-4

Reserpine Sigma 50-55-5 200-047-9

Eserine

(Physostigmine)

hemisulfatesalt

Sigma 64-47-1 200-585-4

BufferComponents.

BufferComponent

Company

CAS#

o-PhosphoricAcid 85% FisherScientific 7664-38-2

Sodiumphosphate,

monobasic, monohydrate

J.T. Baker 10049-21-5

Sodium phosphate,

dibasic,

anhydrous

J.T. Baker 7758-11-4

Citricacid,anhydrous J.T. Baker 77-92-9

Sodiumdihydrogencitrate anhydrous

Fluka Chemica 18996-35-5

Sodium hydrogencitrate sesquihydrate

Aldrich Chemical 6132-05-4

Methanol J.T. Baker 67-56-1

Capillary

Electrophoresis Reagents.

CE Reagent

Company

Part#

1.0 N Sodium Hydroxide Solution for HPCE

Hewlett Packard 5062-8576

0.1 N Sodium Hydroxide Solutionfor HPCE

Hewlett Packard 5062-8575

Waterfor HPCE Hewlett Packard 5062-8578

Instrumentation

.

Capillary

Electrophoresis

Hewlett

Packard

HP

3D-CE,

Model

#

G1602A,

Serial

#

3546G00733

Computer:

Hewlett

Packard Vectra

XMseries3 5/90.

Control

Program:

Hewlett Packard HP

3D-CE Chemstation.

Revision A.05.04

[273]

Labline Environmental Orbital

Shaker.

Rotary

Evaporator:

Labconco

Centrivap

Concentrator.

Catalog

#

78 1 00-00

Labconco Cold Trap.

Catalog

# 781 10-00

Fisher Scientific Vacuum Pump. Maxima C Plus. Model M4C.

pHMeter:

VWR Scientific. Model8005

Beckman Combination Electrode 39846.Lot 59098

Experimental Supplies.

Capillary:

Polymicro Technologies. 25pm Internal Diameter Fused

Silica

_capillary,

360 pm Outer Diameter Polyimidecoating. Part

#:

200001 1

Syringes:

Becton DickinsonandCompany: 10cc _syringe,2.5 cc syringe

Filter discs:

Acrodisc,

sterile syringe

filter. Pore

size:

0.2pm,

Diameter: 25mm. HT Tuffryn

membrane.

Product

#4142

Commercially

Available Products:.

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Yohimbe Powermax2000- _{50 capsules}

Pure

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Pure Yohimbe Bark Extract. Maximum Potency. Fast Acting. ExtractofYohimbe Bark: 2000mg/2ml

General Nutrition

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Men's Yohimbe 451-_{60 Capsules}

Standardized Herbal Support Preparation.

Yohimbe Bark Extract(Pausinystaliayohimba):451 mg/Capsule

(2% Yohimbine Alkaloid=

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Yohimbe Bark Extract.

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Powerman

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The All Night

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Yohimbe

Bark Extract. Yohimbe Bark

Extract:

225 mg/Tablet

Standardized for

_{4.5 mg}yohimbine alkaloids

FactorialandFractional Factorial Design.

Statistical Design Software.

Minitab 12 for Windows. Release 12.21. Minitab Inc. 1998

Methods.

Experimental Design.

Run voltage,

temperature,

buffer composition, buffer pH, buffer ionic strength,

injection

type,

injection setpoint, organic modifier _{concentration,} and effective column

lengthcomprisedthe

factors

chosenfor fractional

factorial design

one (Table 4).

A

two-level,

one-quarterfractional factorialexperimentaldesignatresolutionfour

was compiled_{using design}generatorsprovided

by

Minitab(Figure 6). Fourcenterpoints

Table4. FactorlevelschosenforFFD1. Low levelrepresented

by

_-,high levelrepresented

by

+,

centerpoints represented

by

0. FactorsDandFbasedonfactorsCand_{E,respectively}(i.e.whenCis-,

correspondingtocitrate

buffer,

D+_{is 6. WhenCis +,D}+correspondstophosphatebufferat pH8).

Factor

>

A B C _{D(C-) D(C+)} E _{F(E-) F(E+)} G H J

Level T kV Buffer Citrate PH

Phosphate

PH

Injection

Electro-kinetic Inj(kV)

Pressure Inj(mbar)

Column Length

Methano (%)

Ionic Strength

25 10 Citrate 3 3

Electro-kinetic

5 20 25 0 0.01

+ 35 20 Phosphate 6 8 Pressure 15 50 75 10 0.1

0 30 15 X 4.5 6.5 X 10 35 50 1 0.05

G=ABCD H=ACEF J=CDEF

Figure6. Designgeneratorsfor factorialdesignone. Generated

by

Minitab Statistical Software.

were added to complete the

design,

_{resulting in 68} experimental runs performed

in

duplicate (Table 5).

[image:40.537.47.489.100.313.2]

[image:41.537.114.431.106.688.2]

Table 5. DesignMatrix forthefactorial designone. Designgenerators

G=ABCD,

_H=ACEF,andJ=CDEF.

The leveloffactor G for_anyrunis determined

by

thelevelsoffactors_{A, B,}

C,

andDforthatrun.

(example:runthree._A=-,

B=+, C=-, D=-, E=-,

F=- _{Thereforethelevel}offactor G=_ABCD=

(-)(+)(-)(-)

=

-,leveloffactorH=_ACEF=

(-)(-)(-)(-)

=

+)

Run# A B C D E F G H J

1 - - - + + +

2 + - - - +

3 - ₊ - - - ₊ +

4 + + - - - - ₊ - ₊

5 - - ₊ - - -

-6 + - + - - - _{+ +}

-7 - _{+ +} - - - ₊ -

-8 + + + - - - - +

-9 - - - ₊ - - - +

-10 + - - + - - ₊ -

-11 - ₊ - ₊ - - _{+ +}

-12 + + - ₊ - - - -

-13 - - _{+ +} - - + - +

14 + - _{+ +} - - - _{+ +}

15 - _{+ + +} - - - - +

16 + + + + - - _{+ + +}

17 - - - - + - + -

-18 + - - - ₊ - - ₊

-19 - + - - + - - -

-20 + + - - ₊ - _{+ +}

-21 - - ₊ - + - - + +

22 + - + - + - ₊ - ₊

23 - ₊ + - + - + + +

24 + + + - + - - - +

25 - - - + + - - - +

26 + - - + + - _{+ + +}

27 - + - + + - ₊ - ₊

28 + + - + + - - + +

29 - - ₊ + + - + +

-30 + - _{+ + +} - - -

-31 - + + + + - - +

-32 + + + + + - ₊ -

-33 - - - _{+ +} -

-34 + - - - - + - +

-35 - + - - - + - -

-36 + + - - - + + +

-37 - - + - - + - _{+ +}

38 + - + - - _{+ +} - ₊

39 - + + - - + _{+ + +}

40 + + + - - + - - ₊

41 - - - ₊ - + - - +

42 + - - ₊ - _{+ + + +}

43 - ₊ - ₊ - + + - +

44 + + - ₊ - ₊ - _{+ +}

45 - - _{+ +} - _{+ + +}

-46 + - _{+ +} - ₊ - -

-47 - _{+ + +}

- + - +

-48 + + + + - _{+ +} -

-49 - - - - _{+ + + + +}

50 + - - - _{+ +} - - +

51 - ₊ - - + ₊ - _{+ +}

52 + + - - _{+ + +} - +

53 - - + - _{+ +} - -

-54 + - ₊ - + + _{+ +}

-55 - _{+ +} - _{+ + +} -

-56 + + + - _{+ +} - ₊

-57 - - - _{+ + +} - ₊

-58 + - - _{+ + + +} -

-59 - ₊ - _{+ + + + +}

-60 + + - _{+ + +} - -

-61 - - _{+ + + + +} - +

62 + - + + + + - _{+ +}

63 - + + + + + - - +

64 + + + + + + + + +

65 0 0 0 0 0 0 0 0 0

66 0 0 0 0 0 0 0 0 0

67 0 0 0 0 0 0 0 0 0

68 0 0 0 0 0 0 0 0 0

The secondary investigation focusedonthree

factors

attwo

levels,

buffer

_pH,

buffer

ionicstrength,andrunvoltage(Table 6).

A full factorial designof

23

experimentalruns and an additionaltwocenterpoint

runswerecompleted,

totaling

tenrunsin duplicate (Table 7).

Table6.Factorlevelschosenfor factorial designtwo.(Low levelrepresented

by

high levelrepresented

by

_+,centerpointsrepresented

by

0)

Factor A B

Level PH Voltage Ionic Strength

4 10 0.01

+ _{5 20 0.1}

[image:43.537.161.379.81.273.2]

0 4.5 15 0.05

Table 7. Designmatrixforfactorialdesigntwo.

Run# Level A Level B Level C

1 - -

-2 + -

-3 - ₊

-4 + +

-5 - - ₊

6 + - +

7 - _{+ +}

8 + + +

9 0 0 0

10 0 0 0

[image:43.537.165.369.343.503.2]

Preparation ofBuffers for

Capillary

Electrophoresis.

Citrate and Phosphate

buffers

employed

in

this investigation were prepared

utilizing the

Henderson-Hasselbach

(Equation

4)

and ionic strength (Equation

5)

equationstowarda

known

pH and

ionic

strength

(Scheme

4).

pH=

pKa+

log

_[conjugate

base]

[conjugate acid]

I=_0.5Z

(cjZj2)

Buffers that

did

not measure to the calculated pH were recalculated with a

correction

factor

equalto thatofthedeviation (i.e. abufferwith a measured pH of5.2 has

a deviation of 0.2 pH units. 0.2 is the correction

factor.

The buffer is recalculated

towardsapH of

4.8.)

All buffersolutions were

filtered

througha0.2 pmAcrodisc syringe

Citratebuffer: pH=₅

.00,1

=_{0. 1}

0,

pKa2citrate=_4.76

[H2A]

=

C6H707"

[HA-]

=

C6H607+

0.10=_0.5

(([H2A"](12)

+

([Na,+](12))

+

([HA=](22)

+

2[Na2+](l2)))

Setting [H2A]

=

[Naf]

=_x

and

[HA=]

=

[Na2+]

=

y

0.10=_0.5

(((x)

+

(x))

+

((4y)

+

(2y)))

Solving

forx

x=

0.10-3y

6

The Henderson-Hasselbachequationfortheseconditionsis

5.00=_4.76+

log (y/x)

Solving

fory/x yields

10-24

=

(y/x)

Substituting

Equation6 forx

1.738=_y/(0.10

-3y)

Solving

for yyields

y=_{0.028 M}=

[HA=]

Substituting

y into Equation 6

x=_{0.016 M}=

[H2A']

Scheme 4: BuffercalculationsforcitratebufferpH_5.00,1= 0.10.

Preparation

of

Standards

for

Capillary

Electrophoresis.

Yohimbineand

Eserine Standard (Factorial

_{Design One).}

A measured amount of yohimbine was _{quantitatively} transferred to a 10 mL

volumetric

flask.

Buffer

corresponding

to the proper pH and ionic strength

for

the

experimental run was addedtovolume and mixedtoyield a2000ppm standard.

A measured amount of eserine was _{quantitatively} transferred to a 10 ml

volumetric

flask.

Buffer corresponding to the proper pH and ionic strength for the

experimentalrunwas addedtovolume andmixedtoyielda2000ppm standard.

Equal parts 2000 ppm yohimbine standard and 2000 ppm eserine standard were

added to a 10 ml conical plastic graduated centrifuge tube and vortexed to yield a

standard of 1000 ppm in both yohimbine and eserine. The standard solution was then

filteredthrougha0.2pmAcrodisc syringefilter

disc.

Yohimbineand EserineStandard (Factorial Design Two).

Deionized ultra filtrated water was employed to utilize the _stacking effects that

occur when a buffer oflower ionic strength than the separation

buffer is

present in the

injectionvial (50). Ameasured amountof yohimbine was _{quantitatively} transferred to a

10 ml volumetricflask. Deionizedwater wasaddedtovolume and mixedtoyielda2000

ppm standard.

A measured amount of eserine was _{quantitatively} transferred to a 10 ml

volumetricflask. Deionizedwaterwas addedtovolume and mixedto yield a

2000

ppm

standard.

Equal parts 2000 ppm yohimbine standard and 2000 ppm eserine standard were

added to a 10 mL conical plastic graduated centrifuge tube and vortexed to yield a

standard of 1000 ppm

in both

yohimbine and eserine. The standard solution was then

filtered

througha0.2 pm

Acrodisc

syringe

filter disc.

MethodsofExtraction.

ExtractionofYohimbine from Liquid Products.

Portions of 1 ml of ethanol based

liquid

products were evaporated with a

centrivap concentrator. _{The resulting} solids were reconstitutedwithdeionized water and

filtered with a 0.2 pm Acrodisc syringe filter. An aliquot ofthe filtrate was combined

with an aliquotofinternalstandardandbufferand

injected

ontothecolumn.

Waterbasedproducts werefilteredwith a0.2 mmAcrodisc syringe

filter,

and an

aliquot ofthe filtrate was combined with an aliquot of

internal

standard and

buffer

and

injectedontothecolumn.

ExtractionofYohimbine from Solid Products.

Capsules were broken open and the cellulose _casing

discarded.

Portions

ofthe

powdersremovedwereutilized

for

thisinvestigation. Tabletswere crushed with a mortar

andpestle, andtheresultant powder utilized

for

this

investigation.

Extraction

Method

One.

Portions of0.1 gram of

bark

powder(0.1 gram of_product) was wet and brought

tovolume

in

a 10 ml volumetric

flask

with

deionized

water. _{This slurry} wasagitated for

20 minutes at room temperature. The contents were then filtered through a 0.2pm

Acrodiscsyringe

filter,

and an aliquot ofthe

filtrate

was injectedontothe columnwith an

aliquot of an

internal

standard and

buffer.

Extraction Method Two.

Portionsof0.05 gramof

bark

powder (0.5 gram of_product) was wet andbrought

tovolume in a 10mlvolumetric flaskwithdeionizedwater. This slurry wasagitatedfor

20 minutes at room temperature. The contents were then filtered through a 0.2 mm

Acrodisc syringe

filter,

and an aliquot ofthe filtratewas injectedontothe columnwithan

aliquotof aninternalstandard andbuffer.

Extraction Method Three.

Portions of0.1 gram ofbarkpowder (0.1 gram of_product) was wet and

brought

tovolume ina 100mlvolumetricflaskwithdeionizedwater. This slurrywastransferred

to a 1 L Erlenmeyer flask and agitated for 60 minutes at

60

C in a Labline

environmental orbital shaker. Thecontents were allowed_{to cool,}andthe

liquid decanted

and filtered through a 0.2pm Acrodisc syringe

filter.

An aliquot of the

filtrate

was

injectedontothecolumn withanaliquotof aninternalstandard and

buffer.

Extraction

Method

Four.

Portions

of0.1 gram of

bark

powder (0.1 gram of_product) was wet and brought

tovolumein a 100 ml volumetric

flask

with

deionized

water. _{This slurry}was transferred

to a 250 ml

Erlenmeyer flask

and sonnicatedfor 15 minutesat room temperature. The

contents were allowedto coolto room

temperature,

and the liquid decanted and filtered

through a 0.2pm Acrodisc syringe

filter.

Analiquot ofthe

filtrate

was injectedonto the

column with an aliquot ofan

internal

standard and

buffer.

Extraction Method Five.

Portions of0.1 gram ofbarkpowder(0.1 gram of_product) was wet andbrought

to volume in a 10 ml volumetric

flask

with 10 ml of 95% ethanol. _{This slurry} was

agitated

for

20 minutes atroom temperature. The contents were then

filtered

through a

0.2 pm Acrodisc syringe filter. The ethanol was evaporated with a _rotary evaporator

concentrator, andthe _remaining powderreconstituted

into

3 ml ofdeionizedwater. The

solution was thenfilteredthrough a0.2 pm Acrodisc syringe

filter,

andan aliquotofthe

filtrate

wasinjectedontothecolumn withan aliquot ofan

internal

standard and

buffer.

Extraction Method Six.

Portions of0.1 gram ofbarkpowder (0.1 gram of_product) was wet and

brought

tovolume

in

a 10 mlvolumetric

flask

withchloroformand added toa 125 ml

separatory

funnel.

It was equilibrated with 10 ml of deionized water, and the organic

layer

collected. The aqueous layer was washed with a portion of 10 ml chloroform. The

organic

layers

werecombined, and

filtered

through a0.2 pm

Acrodisc

syringe

filter.

The

chloroform was removed with a _{rotary evaporator,} and the resultant solids reconstituted

into

3 ml of

deionized

water.

The

solution was

filtered

through a 0.2 pm Acrodisc

syringe

filter,

and an aliquot was

injected

onto the column with analiquot of aninternal

standard and

buffer.

Extraction Method Seven.

Solid

phase extraction was performed on an Alltech reverse phase _{900 mg}

Cis

solid phase extraction cartridge. The cartridge was first conditioned with 3 ml of

methanol, followed

by

3 ml ofdeionized water. 1 ml ofsample dissolved in deionized

water (0.05 g/ 10 ml) was applied. A 5 ml portion of water was pushed through the

column, and collected as a fraction. 3 ml

fractions

of 10% methanol in _water, 50%

methanol in water, 75% methanol

in

water, and 100% methanol were collected

successively. The methanol was removed from these fractions with a _centrivap

concentrator. The resultant solids were reconstituted with 1 ml deionized _water, and an

aliquot wasinjectedontothecolumn with an aliquotof aninternal standard andbufferto

determine

thetargetfraction.

Extraction Method Eight.

Solid phase extraction was performed on an Alltech reverse phase 300 mg

Ci8

solid phase extraction cartridge. The cartridge was

first

conditioned with 3 ml of

methanol, followed

by

3

ml ofdeionized water. 1 ml of sample

dissolved in deionized

water (0.05 g/ 10 ml) was applied. A 3 ml portion of water was pushed through the

column, and collected as a fraction. 1 ml

fractions

of 10% methanol in _water, 50%

methanol

in

_water, 75% methanol

in

_water, and 100% methanol were collected

successively. The methanol was removed

from

these

fractions

with a _centrivap

concentrator. The resultant solids were reconstituted with 1 ml

deionized

_water, and an

aliquot was

injected

ontothecolumn with an aliquot of aninternal standardand

buffer

to

determine

the target

fraction.

Extraction Method Nine.

Solid phase extraction was performed on an Alltech reverse phase _{900 mg}

Cig

solid phase extraction cartridge. The cartridge was first conditioned with 3 ml of

isopropanol,

followed

by

3 ml ofdeionizedwater. 1 mlofsample dissolved in

deionized

water (0.05 g/ 10 ml) was applied. A 5 ml portion ofwater was pushed through the

column, andcollected as a

fraction.

Two successive 3 ml

fractions

of 100% isopropanol

were collected, followed

by

twosuccessive 3 mlfractionof2% ammonium

hydroxide

in

isopropanol. The isopropanol was removed from 1 mlportions ofthese

fractions

with a

centrivap concentrator. The resultant solids were reconstituted with 1 ml

deionized

water,and an aliquotwasinjectedontothecolumn with an aliquotofaninternal standard

andbuffertodeterminethe target

fraction.

Extraction Method Ten.

Solid phase extraction was performed on an Alltech reverse phase 300 mg

Cig

solid phase extraction cartridge. The cartridge was first conditioned with 3 ml of

isopropanol,

followed

by

3 ml ofdeionizedwater. 1 ml of sample

dissolved in

deionized

water (0.05 g/ 10 ml) was applied. A 3 ml portion of water was pushed through the

column, and collected as a

fraction.

Two successive 1 ml

fractions

of100% isopropanol

were_collected,

followed

by

twosuccessive 1 ml

fraction

of2% ammoniumhydroxide

in

isopropanol.

The isopropanol

was removed

from

these

fractions

with a _centrivap

concentrator. The resultant solids were reconstituted with 1 ml deionized _water, and an

aliquot was

injected

onto thecolumn with an aliquotof an internalstandardandbufferto

determine

the target

fraction.

Optimized

Extraction

Conditions

for Commercial Samples.

Theprotocol of extraction

five

was expandedforcontinuedinvestigation. Sample

mass andethanolvolumewere varied

between

highandlow levelsto

determine

the most

efficient extraction conditions. Thefinal conditions

for

real sample analysis

involved

the

use of 0.05 g sample dissolved into 10 ml of95% ethanol in a volumetric

flask.

The

ethanol was then decanted and filtered through a 0.2 mm Acrodisc syringe filter. 1 ml

aliquots ofthis _slurry were evaporated with a _{Labconco rotary} concentrator (60 C * ₄₅

min). Theresultantsolids werereconstituted

into

1 ml ofwarm

deionized

_water,

filtered

througha0.2mm Acrodisc syringe

filter,

and an aliquot

injected

_along with avolume of

eserinestandardandbuffer.

Instrumental.

Conditioning

ofthe

Capillary

Column.

New capillary columns were preconditioned _utilizing a standard method. A 45

minute

flush

with 1.0