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Journal of Ana lytical Toxicology, Vol. 20, O ctobe r 1996

I d e n t i f i c a t i o n o f U r i n a r y B e n z o d ia z e p in e s a n d t h e i r

M e t a b o l i t e s : C o m p a r is o n o f A u t o m a t e d H P L C a n d

G C M S a f t e r Im m u n o a s s a y S c r e e n in g o f C l i n ic a l

S p e c i m e n s

J imm ie L . Va lent ine , Ro sa lyn M idd le ton, and Ca ro lyn Sparks

Dep ar tments o f Pediat r ics and Pharm acology, Sect ion on Pediat r ic Clin ical P harm acology, Univers i ty of Arkansas for Me dica l

Sciences and Toxicology Lab oratory o f Arkansas Chi ldren s H ospi ta l , L i t tle Rock , Arkansa s 7220 2-3591

I b s t r a c t

An au tomated h igh -pe r fo rmance l i qu id ch romatog raph ic me thod ,

benzod iazep ines by REM EDi HS, was used to ana l yze

benzodiazepines and the i r metabol i tes after 13-g lucuronidase

hydro l ys is o f 1 -m L u r ine s pec imens f rom the fo l l ow ing : 924 c l i n i c

and hosp i ta l pa t i en ts whose spec imens had p rev ious l y been found

to be p resumpt i ve l y pos i t ive us ing e i the r EM IT o r Tr iage

immun oassay me thodo log ies and 128 i nd iv idua ls whose spec imens

had sc reened nega t i ve by EM IT d .a .u . M REM EDi ana l yses d id no t

co r re la te w i th the imm unoassay resu lts in 136 o f the pos i t ive an d

th ree o f the nega t i ve u r ine spec imens . Gas ch romatog raph ic -mass

spec t romet r ic (GC-M S) con f i rma to ry a na l yses were pe r fo rmed on

these d isco rdan t spec imens us ing 3 mL ~-g lucu ron idase -

hyd ro l yzed u r i ne fo ll owed by ex t rac t ion w i th

chloroform -isopropano l (9 :1) and der ivat izat lon wi th

N,O -bis( tr ime thyls i ly l) t r if luoroacetam ide. Tw o benzo diazepines,

f lun i t razepam and c lonazepam , and the i r 7 -amino m e taho l ites

were ana l yzed w i thou t p r i o r de r iva t i za ti on . The ana lyses

e s ta bl is h e d 8 7 c o n c o rd a n c e b e tw e e n R E M E D i a n d GC - M S

versus 13 concordance w i th immunoa ssay fo r the subse t .

GC -MS ana lys is o f these 142 spec imens demons t ra ted two reasons

fo r t h e n o n c o n c u r re n c e b e tw e e n R E M E D i a n d E M IT : E M IT h a d

g iven e i the r fa l se -nega t i ve o r fa l se -pos i ti ve resu l ts and EM IT had

g iven a pos i ti ve resu l t even though the de te rmined metabo l i tes

were be low the 200-ng /mL cu to f f fo r the imm unoassay and the

80-ng /mL cu to f f fo r R EM EDi . A to ta l o f 23 spec imens were found

to con ta in on l y Io razepam by REM EDi and GC-MS, 15 o f wh ich

had been sc reened by Tr i age . A reeva lua t ion o f these 23 spec imens

by EM IT d .a .u , demons t ra ted tha t 11 were pos i t ive . T h i s f i nd ing

was i n con t ras t to p rev ious repor ts tha t EM IT w i l l no t de tec t

Io razepam g lucu ron ide i n u r ine . An unexp ec ted f i nd ing was the

REM EDi i den t if ica t i on and subsequen t GC -MS con f i rma t ion o f

7 -amino f lun i t razepam , a u r i na ry me tabo l i te o f f lun i t razepam tha t

is not avai lab le in the U ni ted S tates and that rep resented i l l i c i t use

by fou r pa t i en ts . A d i s t inc t advan tage o f REM EDi p roved to be i ts

capab i l it y i n i den t ify ing demo xepam, a m a jo r me tabo l i te o f

ch lo rd iazepox ide ; G C-M S ana lys i s cou ld no t de tec t th i s me tabo l i te

because o f i ts the rma l decom pos i t ion to no rd iazepam. To fu r the r

eva lua te the spec i fi c ity o f REM EDi , we conduc ted GC -MS ana lyses

in a random fash ion on 55 add it iona l nond isco rdan t u r i ne

Author to whom correspondence should be addressed.

specim ens tha t were ident i f ied as e i ther posi t ive o r negat ive, as

we l l as 22 spec imens i den t if ied as con ta in ing 7 -aminoc lonazepam

by REM EDi . Concur rence was observed be tween the two methods

fo r a l l spec imens , w i th the excep t i on o f one apparen t fa l se pos i ti ve

fo r u . -hyd roxya lp razo lam by REME Di . The rep ro duc ib i l i t y o f the

REM EDi me thod was found to be exce l len t ; i t was assessed by

compa r ing resu l ts o f 266 spec imens tha t were rep rocessed i n

d i f fe ren t ba tches and fo r know n ca l ib ra to rs and con t ro l s a l so

p rocessed w i th each ba tch . S tudy resu l ts demon s t ra ted tha t the

au tomated REM EDi assay fo r u r i na ry benzod iazep ines and the i r

me tabo l i tes was comparab le w i th G C-MS b u t had d i s t i nc t

advan tages ove r G C-M S because o f the fo l l ow ing reasons :

s impl ic i ty o f the assay, less t ime requi red for a nalyses, and

provis ion of addi t ional in formation conce rn ing the parent

benzod iazep ine .

n t r o d u c t i o n

B e n z o d i a z e p i n e s a r e w i d e l y p r e s c r i b e d d r u g s i n b o t h t h e

U n i t e d S t a te s a n d o t h e r n a t i o n s , a n d t h e i r a v a il ab i li ty d e p e n d s

o n l o ca l r e g u l a t i o n a n d , i n s o m e i n s t a n c e s , i ll e ga l d i s t r i b u -

t i o n . t n t h e U n i t e d S t a te s , t h r e e g r o u p s o f b e n z o d i a z e p i n e s

a r e a v a il ab l e b a s e d o n t h e i r c h e m i c a l s t r u c t u r e s ( 1) ( F i g u r e 1 ) ,

n a m e l y , 1 , 4 - b e n z o d i a z e p i n e s , d i a z o l o b e n z o d i a z e p i n e s , a n d

t r i a z o l o b e n z o d i a z e p i n e s ( T a b le I ). I d e n t i f i c a t i o n o f t h e a d m i n -

i s t e re d b e n z o d i a z e p i n e b y a n al y si s o f a u r i n e s p e c i m e n i s c o m -

p l i c a te d b y t h e f a c t t h a t m o s t o f t h e s e d r u g s u n d e r g o e x t e n s i v e

p h a s e I a n d I I m e t a b o l i s m a n d o n ly m i n o r a m o u n t s o f t h e

u n c h a n g e d d r u g a r e e x c r e te d a n d b y t h e f a c t th a t s o m e o f t h e

1 ,4 - b en z o d ia z e p in e s h a v e c o m m o n m e t a b o l i c p a t h w a y s ( F i g u r e

2 ) . F u r t h e r c o m p l i c a t i n g t h e a n a l y t i c a l i d e n t i f i c a t i o n i s t h e

f a c t t h a t m a n y m e t h o d o l o g i e s c a n n o t d i s t i n g u i s h b e t w e e n t h e

v a r i o u s m e t a b o l i t e s .

M o s t la b o r a t o r ie s u s e s o m e t y p e o f i m m u n o a s s a y s c r e e n f o r

d e t e c t i n g t h e p r e s e n c e o f a b e n z o d i a z ep i n e ; e x a m p l e s a r e

r a d io i r nm u n o a s s a y , e n z y m e m u l t ip l i e d i m m u n o a s s a y ( E M IT ~

f l u o r e s c e n c e p o l a r i z a ti o n i m m u n o a s s a y , l a te x a g g l u t i n a t i o n

( O N - Tr a c| i m m o b i l i z e d m o n o c l o n a l a n t i b o d i e s ( T ri a ge | o r

e n z y m e - l i n k e d i m m u n o s o r b e n t a ss ay (S I N G L E S T E P ' ) .

4 6 Reproduction photocopying)of e ditorial contentof this ou rnal s prohibitedwithout publisher'spermission.



Journal of Analytical Toxicology, Vol. 20, October 1996

Regardless of the screening technolog y used, many reports of

false-positive and false-negative results for the benzodiazepines

have appeared 2-10). Another comm only used screening tech-

nique, thin-layer chrom atograph y TLC),was reported to pro-

duce false-negative results for some benzodiazepines 11), and

furtherm ore, the individual m etabolites cannot be identified

because conversion to aminobenzophenones is required for

visualization. Therefore, confirm ation of scr eening technique s

is required to assist in developing a definitive clinical diagnosis

or to assess forensic implications.

A numbe r of confirmatory techniques are widely used for the

benzodiazepines, such as high-performance liquid chro-

matog raphy HPLC) followed by UV detect ion 12,13), gas

chromato graphy-ma ss spectrom etry GC-MS) 14-17), or gas

chromatography-electron capture detection GC-ECD)

18,19). The use of GC for the separation of the benzo-

diazepines is lim ited by the requ irem ent for an elevated injec-

tion port temperature; the high temperature transforms a

major metabolite of chlordiazepoxide, demoxepam, into nor-

diazepam, which is a major metabolite of a num ber of 1,4-ben-

zodiazepines 20,21) Figure 2). The decompositio n of this

N-oxide metabolite during eithe r GC-MS or GC-ECD analysis

renders the identification of chlordiazepoxide adminis tration

indistinguishable from other 1,4-benzodiazepines that also

have nordiazepam as a metabo lite 22). An additional compli-

cating factor is that most benzodiazepines taken in therapeutic

amou nts are found in nanogram-per-milliliter concentr ations

in urine, which make it necessary to use electron impact El)

MS with selected ion mon itor ing SIM). Because of the wide

variety of urinary parent benzodiazepines and thei r m etabo-

lites, it is often necessary to limit the number of analytes

detected simultaneously us ing EI-MS with SIM because of the

following reasons: a) differences in chro mat ogra phic and

thermal stability properties that require some analytes to be

derivatized while others are analyzed wit hout derivatization, b)

requirem ents for moni torin g of multipl e ions to ensure selec-

tivity, c) diverse polar and m ass characteristics requ iring dif-

ferent preanalytical work-up and c hromatog raphic conditions,

and d) differences in the d etection limits as a result of the

varying concentratio ns found in urine because of the diverse

dosages used to achieve a therapeutic effect.

Of the potential confirmatory m ethods, TLC and HPLC do

not produce pyrolytic deco mposi tion of the benzodiazepines.

However, as already noted, TLC cannot distinguish between

different mem bers of the class if conversion to benzophenones

is used before chromatography. HPLC was used by a number o f

workers to determine various benzodiazepines in blood

12,23-26) and urine 13,22,27) and can be used for identifi-

cation of demoxepam 22); hence, it permits the assi gnmen t of

chlordiazepoxide as the pare nt drug Figure 2).

The REMEDi HS drug profiling system, an automated

HPLC-based instru ment , is widely used to identify basic and

neutral drugs 28-31). A comm ercial kit columns, reagents,

and software) is now available for the analysis of urina ry ben-

zodiazepines and their metabolites. The utility of this tech -

nology was evaluated with urine specimens from a variety of

clinic and hospital patients that had previously tested positive

by immunoassay and was also evaluated with u rine specimens

from a contr ol group of specimens th at had tested negative for

benzodiazepines. GC-MS analyses were perform ed on all spec-

imens whose results were disparate between the immuno assay

and REMEDi methods. A representative num ber of nondiscor-

dant specim ens that had tested either positive or negative for

various benzodiazepines and their metabolites were randomly

chosen and also tested by GC-MS to further determ ine the

specificity of the REMEDi metho d.

Experimental

Chemkals and reagents Acetone, ethyl acetate, sulfuric

acid, potassium dichromate, sodium acetate trihydrate, and

sodiu m bicarbonate were obtained from Mallinckrodt. Nor-

diazepam, oxazepam, oxazepam-d5, diazepam-d5, lorazepam,

temazepam, 2-hydroxyethylflurazepam, ct-hydroxyalprazolam,

R

B

R I C H ~ N

CI R

G

CI R

Figure 1. Structures of A) 1,4-benzodiazepines, B)

diazolobenzo-

diazepines, and C) triazolobenzodiazepines. See Table I for R groups.

4 7



~-hydroxytriazolam, clonazepam, 7-aminoclonazepam , fluni-

t razepam, and 7-aminof luni t razepam were obta ined f rom

Radian as methanolic solutions containing either 1 mg/mL or

100 IJg/mL. The following benzodiazepines were sup plied by

Roche Pharmaceuticals: midazolam, ~-hydroxymidazolam,

and 4-hyd roxym idazolam. Chloroform, i sopropanol , and

m e tha no l we r e ob t a ine d f r om Bur d ic k Ja c kson , a nd

N,O-bis trimethylsilyl)trifluoroacetamide (BSTFA) was

obtained from Pierce. Chloroform-isopropanol (9:1) was pre-

pared as a volume-to-volume ratio solution. ~-Glucuronidase,

type H2, from Helix pom atia (89,400 units/mL) was from

Sigma. The 50raM sodium bicarbonate solution (pH 11) was

prepared by dissolving 2.1 g NaHCO3 in 500 mL o f reagent-

grade water . The 2M sodium acetate buffer (pH 4.8) was

J o u r n a l o f A n a l y t ic a l T o x i c o l o g y , V o l . 2 0 , O c t o b e r 1 9 9 6

prepared by dissolving 68.05 g sodium acetate trihydrate in

250 mL o f reagent-grade water.

GC-M S internal standard solutions. Two a m pule s o f

oxazepam-d5 each containing 100 pg/mL in methanol w ere

transferred quantitatively to a 100-mL class A volumetric flask

and diluted to vo lum e with methanol to giv e a final concen-

tration of 2 pg/mL. The solution was transferred to a 100-mL

Repipeter (Labindustries) fitted with a fixed 0.5-mL syringe,

and the solution was stored at 5~ when no t in use. The syringe

delivered 0.5 • 0.01 m L (1000 ng) o f the internal standard

solution. The diazepam-ds internal standard solution was pre-

pared in a similar mann er.

Urine specimens an d initial irnrnunoassayscreening. Urine

specimens were obtained from four different medical centers in

T a b l e I . N a m e s o f t h e 1 , 4 - B e n z o d i a ze p i n e s , D i a z o l o b e n z o d i a z e p in e s , a n d T r i a z o lo b e n z o d i a z e p i n e s A v a i la b l e i n t h e U n i t e d

S t a t e s a n d T h e i r P r i m a r y U r i n a r y M e t a b o l i t e s

Parent Metabolite(s) RI R R R R R

1 4 - B e n z o d i a z e p i n e s

Chlordiazepoxide H N H C H ] H ~ O t C I H

Norchlordiazepoxide H NH2 H ~ O t C I H

Demoxepam H H H ~ O t C I H

Diazepam CH] =O H C I H

Nordiazepam H =O H CI H

Oxazepam*w H =O OH CI H

Temazepam*w CH ] =O OH CI H

Prazepam CH2<] =O H CI H

3 - H y d r o x y p r a z e p a m C H 2 < ] =O OH C] -

Nordiazepam , o x a z e p a m . . . . .

Clorazepate H O H ) 2 C O 2 H Cl H

Nordiazepam , o x a z e p a m . . . . .

Lorazepam

* H = O

OH CI CI

Flurazepam

Clonazepam

-(CH2)2N(C2Hs) =O H CI F

2-Hydroxyethylf lurazepam * -(CH2)2NHC2H5OH =O H CI F

Desalkylf lurazepam H =O H CI F

3-Hydroxy-desalkylf lurazepam H =O OH CI F

H =O H NO2 CI

7-Aminoclonazepam H =O H NH2 CI

7-Acetamidoclonazepam H =O H - NHCO CH3 CI

D i a z o l o b e n z o d i a z e p i n e

Midazolam

H H

0c-Hydroxymidazolam OH H

4-Hydroxymidazolamu H OH

T r i a z o l o b e n z o d i a z e p i n e s

Alprazolam

Estazolam

Triazolam

CH] H H

~c-Hydroxyalprazolam CH2OH H H

4-Hydroxyalprazolam CH 3 H H

H H H

4-Hydroxyestazolam f H OH H

CH~ H CI

o~-Hydroxytriazolam CH2O H H CI

4-Hydroxyt r iazolam CH3 OH C l

* Bo l d nam e i nd i c a tes av a i l ab i l i t y as a p res c r ip t i on d rug i n t he U n i t ed S ta tes .

N - o x i d e .

P res en t p red om i nan t l y as t he 3 -g l uc u ron i de .

A l s o av a i l ab l e as t he pa ren t d rug . Me tabo l i s m i s ma i n l y as 3 -g luc u ron i de .

u A m i no r m e tabo l i te .

4 1 8



Journal of Analy t i ca l Tox ico logy Vol . 20 Octobe r 1996

the Lit t le R ock area (Arkansas Children 's Hospital , Baptis t

Medica l Cen ter , and McCle l lan and For t Roo ts Veterans

Adm inistration Hospitals) . A total of 877 spec imen s had been

screened by EM IT technology o n a variety of cl in ical chem istry

a n a l y z e r s ( R o c h e M o n a r c h , O l y m p u s R E P LY , B e c k m a n

Syn chro n CX, and Syva ETS), whereas 37 had b een evaluated

wi th the T r iage techno logy on spec im ens o r ig ina t ing f rom the

emergency de par tmen t o f each fac il ity . The spec im ens were

ob ta ined f rom a var ie ty o f in - and ou t -pa t ien t s whose

a g e

groups range d fro m neona tes to older adults. Additionally, 128

specimens subm itted as part of our forensic urine drug-te st ing

p ro g ra m a n d t h a t h a d b e e n s c r e e n e d n e g a t i v e fo r b e n z o -

diazepines by EMIT d.a.u. on the Syva ETS were also evalu-

a ted . Al l med ica l cen ters u sed the manu fac tu rers ' recom men -

dations fo r analyses and a cu toff of 200 ng/mL . This s tudy was

a p p r o v e d an d j u d g e d e x e m p t f r o m t h e r e q u i r e m e n t f o r

inform ed cons ent by the U niversi ty of Arkansas for Medical

Sciences H um an R esearch Advisory Co mm ittee, and speci-

me ns w ere supplied to the laboratory in a coded fashion.

Preparation of in h ouse calibrators and controls.

The in-

house urine cal ibrators and contro ls were prepared with u rine

collected from laboratory perso nnel . Each lot of urin e col-

lected was show n to be devoid of exogenous benzodiazepines by

bo th EM IT d .a.u , and GC-MS screen ing and had 30 mg sod ium

azide added per 4 L urine. Calibrators were prepared by quan-

t i t a t ive ly t rans fer r ing the con ten ts o f ampules , con ta in ing

e i t h e r 1 m g / m L ( n o r d i a z e p a m , o x a z e p a m , l o r a z e p a m ,

temazepam; on e ampu le per drug) or 100 pg/m L (2-hydroxy-

ethylflurazepam, ~x-hydroxyalprazolam, and r

zolam; 10 ampu les per drug), to a 1-L class A volum etric flask

and d i lu t ing to vo lume wi th u r ine to g ive a f ina l concen t ra tion

of 1000 ng /mL. Sub sequen t d i lu t ions o f th i s s tock were made

wi th the u r ine to g ive , in add i t ion to the 1000 ng /mL ca l i-

brator, o th er cal ibrators w ith con centr at ions of 500, 250, 125,

62.5 , and 31.25 ng/m L. Addit ional ly, ano the r set of cal ibrators

con ta in ing 1000 , 500 , 250 , and 125 ng /mL of th is sam e g rou p

of benzodiazepines bu t w ithou t 2-hydroxyethylflurazepam was

prepared and used in REM EDi analyses. In a s imilar ma nner,

two d i f fe ren t se t s o f ca l ib ra to rs were made con ta in ing the

fo rm e r c o n c e n t r a t i o n s . On e s e t c o n t a i n e d m i d a z o l a m , 4 -

hydroxymidazolam, and a-hydroxymidazolam, and the other

con ta ined c lonazepam, 7 -aminoclonazepam , f lun i t razepam ,

and 7-am inoflunitrazepam. C ontrols were prepared in a similar

m anne r on a d i ffe ren t day and con ta ined on ly oxazepam a t

I C h l o r d i a z e p o x i d e ]

D i a z e p a m 1 ~ _ _

No rc h l o rd i a z e p o x i d e

N o r d i a z e p a m

o r a z e p a t e [

i i

u r a z e p a m l

2 H y d r o x y e th y l flu r a z e p a m

N - D e s a l k y l f lu r a z e p a m ~ N - D e s a l k y l- 3 - h y d r o x y f lt r a z e p a m - ! ~

C l o n a z e p a m , .- - 7 -Am i n o c l o n a z e p a m , -- 7 -Ac e t a m i d o c l o n a z e p a m

D e m o x e p a m [ T e m a z e p a m ] - - - - 1 ~

U

U

[ M i d a z o la m [ ~ 4 -H y d ro x y m id a z o la m .

a - H y d r o x y m i d a z o l a m " - -

4 - H y d r o x y a l p r a z o l a m

A l p [ H y d l pa z o l a m ~ ~ - ro x y a r a z o l a m , ' -

4 -Hy d ro x y t r i a z o l a m

T r i a z o b m

[ , r ~

c t -Hy d ro x y t r i a z o l a m

E s t a z o l a m I 9 - r o x y e s t a z o l a m

- H y d

I

R

N

I

D

A

T

I

N

F i g u r e2 . A s c h e m a t ic o f th e m a j o r m e t a b o li s m o f b e n z o d i a z e p in e s . T h o s e d ru g s c o n t a i n e d w i t h in a b o x a r e a v a i la b l e i n t h e U n i te d S t a te s a n d a r e t h e p a r e n t

a d m i n is t e re d c o m p o u n d . A d a p t e d f ro m S . C . H a r v e y . H y p n o t ic s a n d s e d a t iv e s . n T h e P h a r m a c o l o g ic a l B a s is o f T h e r a p e u t ic s 7 t h e d . A . G . G i lm a n , L .S . G o o d m a n ,

T . W . R a i l, a n d F . M u r a d , E d s . , M a c m i ll a n P u b l i s h in g , N e w Y o r k , N Y , 1 9 8 5 , p 3 4 7 . )

4 9



concentrations of 0, 13 1, and 480 ng/m L. Each urine cali-

brator and control was pipetted as 3.5-mL portions into indi-

vidual storage containers and frozen at -30~ until use. Each

lot of calibrator and controls was validated by GC-MS with

commercially available positive urine control samples con-

taining n ordiazepam (Curtin M atheson Scientific)and through

interlaboratory comparisons in the College of American Pathol-

ogists forensic urine drug and toxicological surv eys for nor-

diazepam, oxazepam, cr and ~-hydroxy-

triazolam.

HP LC analysis of urine specimens. A REMEDi HS drug pro-

filing system w as used for all analyses. The ben zodiazepine

analytical kit, cons isting of three cartridges and five solven ts for

the liquid chromatographic separation, was supplied by the

manufacturer (32). A beta version o f the software (version 5.0)

was also supplied for data analysis. All reagents were p repared

according to the manufacturer 's instructions. A 1-mL aliquot

of the urine specimen had 100 pL each of buffered internal

standard (triazolam and ethyl oxazepam glucuronide) and

hydrolysis reage nt added, after which it was mixed and incu-

bated in an oven at 37~ for 2 h. The mixture was centrifuged

at 5000 rpm for 5 min, and the sup erna tant was injec ted

directly into th e REM EDi system. Each day that specimens

were processed, a set of in-house calibrators (125, 250, 500, and

1000 ng /m L ) a nd c on t r o l s ( 0, 131 , a nd 480 ng /m L of

oxazepam), prepared as already described, were analyzed. Also

analyzed were a manufacturer-suppliedCheck Mix' containing

bromazepam , r oxazepam, lorazepam,

and temazepam and a Limit CheckM containing 7-aminoclon-

azepam, 7-aminoflunitrazepam, dem oxepam, ~-hydroxyalpra-

zolam, cr oxazepam, lorazepam, emazepam ,

and nordiazepam. Thus, a daily batch co nsisted of these cali-

brators and controls plus 41 urine specimens. The manufac-

turer 's claimed cutoff value for seven ben zodiazepine metabo-

lites, nam ely, oxazepam, temazepam, nordiazepam, lorazepam,

(~-hydroxyalprazolam, ~-h ydro xytr iazo lam, and hy droxy-

ethylflurazepam, was 80 ng/mL; for the other metabolites,

namely, demox epam, 7-aminoclonazepam, and 7-am inofluni-

trazepam, the cutoff ranged from 150 to 600 ng/mL .

Analysis of urine specimens with G C-MS . To 3 mL of ur ine

were added 0.5 mL of ei ther oxazepam~ or diazepam-d5

internal standard solution, 100 pL of 2M (pH 4.8) acetate

buffer, and 50 1JL ~-glucu ronidas e follow ed by 5 s of vortex

mixing and incubation for 2 h in a water bath at 55~ This

solution was then coo led to room temperature, followed by

addition of 1 mL 50raM sodium bicarbonate solution (pH J1)

and 3 mL of chloroform-isopropanol (9:1), rotation on a h ema-

tology mixer for 10 rain, and centrifugation at 5~ and 2600

rpm for 10 rain. The l ow er organic layer was removed and

evaporated to dryness und er nitrogen at 50~ To the resultant

residue were adde d 20 IJL ethyl acetate an d 30 IJL BSTFA,after

which th e solution was vortex mixed and h eated in a sand bath

at 75~ for 15 rain. For those specimens th at were to be

analyzed for clonazepam, 7-aminoclonazepam, flunitrazepam,

7-aminoflunitrazepam, and the diazepam-d5 nternal standard,

50 IJL ethyl acetate was add ed, and the solution was vortex

mixed. This solution was transferred to an autosampler vial for

GC-MS analysis. An ethyl acetate blank was processed after

420

J o u r n a l o f A n a l y t i c a l To x i c o lo g y V o l . 2 0 Oc t o b e r 1 9 9 6

each specimen analysis. Eachbatch processed during the study

contain ed a set of six calibrato rs (31.25, 62.5, 125, 250 , 500, and

1000 ng /m L ) a nd t h r e e c o n t r o l s (0 , 131 ,480 ng /m L of

oxazepam). The precision and accuracy in recovery of the two

posit ive controls processed on each batch run gave mean

(range) oxazepam concentrations of 124.5 (122.1-126.8) and

479.1 (47 3.5-484.7) ng/mL. A mean correlation co efficient of

0.95 or greate r was determined for each calibrator set used in

the batch analyses. If the determined concentration was greater

than 1000 ng/mL, the specimen was appropriately diluted and

reprocessed. The l imit of qu anti tat io n was 40 ng /mL for

oxazepam and lorazepam and 65 ng/mL for the oth er benzo-

diazep ines. The limit of detection (LOD) was 10 ng/m L for all

benzodiazepine metabolites except 2-hyd roxyethylflurazepam,

r r r

zolam, 7-aminoclonazepam,and 7-am inoflunitrazepam,which

had an LOD of 35 ng/mL.

GC -MS instrumentation and chromatograph ic conditions.

All ur ine G C-M S analyses we re perform ed on a Hewlett-

Packard model 5972B positive ion electron impact quadrupole

mass spectrom eter interfaced w ith a m odel 58 90 gas chro-

matog raph, a mod el 7673 autosampler, and an HP Vectra

90 MHz Pentium com puter using the D rug-Quant sof tware

(revision A.00.00 ) o r a Hewlett-Packardmodel 5971A positive

ion electron impact quadrupole mass spectrometer interfaced

wi th a m ode l 5890 ga s c hr om a togr a ph , a m ode l 7673

autosampler, and a m odel HP Apollo 9000 series 400 Unixdata

station using Target software (revision C.02.02). The gas

chromatographs were each equipped with a capillary column

(12 m x 0.2-ram i.d., 0.334Jm film thickness) containing an

HP-1 cross-linked methy l silicone gum and operated using a

program with an initial temperature of 70~ (1 rain) that was

the n increased to a final temperature of 290~ at a rate of

15~ the f inal hold was 4.33 min ( total run time, 20

rain). This program was slightly modified for the analysis of

midazolam and its metabolites, namely, a final temperature of

275~ was used (total run time, 19 rain). The carrier gas was

helium; the initial inlet pressure was 8.99 psi/min, followedby

a prog ram of 14.5 psi (0.01 rain) and a final pressu re of 9.0 psi

(flow rate, 1.15 mL/min) for 20 rain. The injector port was

operated at 250~ and fitted wit h a Merlin septum (HP-5181-

8839) and a g lass mixing c ham ber assemb ly (HP-19251-60504)

containing a 10-ram silane-treated glass wool plug (HP-5080-

8764) that w as 30 mm from the end next to the gold-plated seal

(bottom injector plate). The glass m ixing chamber assemb ly

was changed daily and cleaned as previously eported by Valen-

tine e t al. (33). The gold-plated seal was changed at the time o f

column installation an d ion sou rce cleaning. The mass spec-

trometers w ere autotu ned d aily with perfluorotributylamine,

were operated in the SIM mod e with a 50 m s dwell time, and

were set at 400 V gre ater than th e multiplier value obtained in

the daily tune. Specimens were analyzed monitoring the fol-

lowing retentio n times and ions (where q is the quan titative

ion): nord iazepam, 12.6 rain, m / z 341 q) , 342, and 343;

oxazepam-d5, 13.3 min , m /z 433 (q), 435, and 436; oxazepam,

13.3 rain, m / z 429 q) , 430, and 431; lorazepam, 13.9 min,

m / z 429 q), 430, and 431; temazepam, 14.9 rain, m / z 343 q),

345, and 372; midazolam, 14.3 min , m/z 310 q), 312, and 325;



Journal of Analy t i ca l Tox ico logy Vol . 20 Octobe r 1996

2-hydroxyethylflurazepam, 14.7 min,

m / z

288

q ) ,

287, and

389; 4-hydroxymidazolam, 15.0 rain ,

m / z

398

q ) ,

399, and

400; ~-hydroxymidazolam, 15.2 rain ,

m / z

310

q ) ,

398, and

413; ~-hydroxyalprazolam, 16.6 rain ,

r n / z

381

q ) ,

383, and

396; r 17.4 min ,

m / z

415

q ) ,

417, and 430;

diazepam -ds, 13.5 min,

r n / z

261

q ) ,

287, and 289; clonazepam,

15.7 min ,

m / z

314

q ) ,

315, and 280; 7-aminoclonazepam, 15.5

min ,

m / z

285

q ) ,

256, and 257; flunitrazepam, 14.8 min ,

r n / z

312

q ) ,

285, and 2 86; and 7-aminoflunitrazepam, 14.6 m in,

m / z

283

q ) ,

255, and 2 54. A ratio of qual ifier ions to quan ti-

tat ive ion of • was used for al l analyses. The auto sam pler

was set to perform five pu m ps o f the syringe before drawing

2 ~L for in jection. A viscosi ty delay of 1 w as used alon g with ten

washes of ethy l acetate. Sam ples were injected in a spl i tless

mode wi th a pu rge-on t ime o f 2 m in fo l lowed by a 20 :1 sp l it .

Results and Discussion

A total of 924 urine specim ens that w ere presumptively pos-

i t ive for benzodiazepines usi ng ei th er the EM IT or Triage

m e t h o d o l o g i e s we re e a c h e v a l u a t e d b y R E M E Di . Of t h i s

numb er , 788 (85 .3 ) spec imens were found by REMEDi to

con ta in one o r more benzod iazep ines o r the i r metabo l i t es ,

and 136 (14.7 ) we re found to be negative. This lat ter d iscor-

dan t g rou p o f spec imens was subm i t ted to G C-M S analys is .

Fifty-s ix were foun d to be negative; 55 co ntained one or m ore

benzodiazepines and their metaboli tes , each of which were

below the 80-ng/mL cutoff specified by the manufacturer; eight

spec imens con ta ined oxazepam above the 80-ng /mL cu to ff

(mean, 142.1 ng/mL; range 93.5-213.7 ng/mL); two specimens

con tained temazepam at levels of 138.6 and 183.7 ng/mL; and

f ive spec imens con ta ined tem azepa m a t l eve l s g rea ter tha n

the 80-ng/mL cuto ff but with values tha t appeared to be art i -

factual because th e qualifier ions were greater tha n the •

perm it ted by the procedure. Reanalysis of these lat ter speci-

mens gave iden t ica l resu l t s and h in ted tha t an in te r fer ing

com pon ent not identified in the s tudy was responsible for the

result . A dditionally , there w as insufficient spec ime n to per-

form GC -MS confirm ation on five specimens, and five speci-

mens could not be sat isfactori ly analyzed by GC-M S because of

fai lure to extract the internal s tandard. Reanalysis of these

lat ter specimens, up to thre e additional t ime s, gave identical

resu l ts , wh ich suggested tha t some end ogenous cons t i tuen t in

the u r ine mat r ix was p reven t ing the in te rna l s tandard f rom

being extracted or, al ternately , was pr eve nting sat isfactory

derivatization.

An additional 128 urine spe cim ens that were negative for

benzodiazepines using the E MIT d.a.u , me thodolog ywere also

evaluated by REMEDi; only thre e s pecim ens were found to be

pos i t ive , con ta in ing 7 -aminoclonazepam, ~-hydroxyalp ra-

zolam, and demoxepam, respect ively . These three specimens

were also evaluated by GC-MS, and the REMEDi findings were

validated by the identificat ion of nordiazepam.

To fur ther evaluate the finding s of REMEDi, we pe rform ed

three addit ional s tudies. F irs t , 55 of the spe cime ns tha t had

been e i ther pos i tive o r negat ive by immu noassay and con-

firme d ei ther posi t ive or negative by REMEDi were rand om ly

se lec ted fo r GC -MS analysi s. These ana lyses dem ons t ra te d

con curre nce for all 31 REM EDi-posi tivespecim ens evaluated.

For the 24 negative spec imens, on ly one nonco ncurren ce was

no ted , namely , one spec ime n tha t co n ta ined r

zolam by REMED i could no t be conf irmed by GC-M S. Second,

266 of the original immunoassay-posit ive specimen s we re pro-

cessed us ing the same exper imen ta l co nd i t ions on subsequen t

days to evaluate the reproducibility of the REM EDi assay (i.e.,

would the method give the same resul ts on a different occa-

sion?). This s tudy dem onstra ted only s ix (2 .2 ) nonid entica l

resu l ts , mo s t o f wh ich oc curred when , as shown by GC-MS

analysis , one or mo re benzodiazepine metabolites we re at or

near the manufacturer 's s tated cutoff. Third , during the pro-

cessing of 23 different batch analyses on the REME Di instru-

me n t , a se t o f in -house ca lib ra tors and c on t ro l s and one each

of the m anufac tu rer -supp l ied ca lib ra tors and c on t ro l s were

processed. All 23 negative c ontr ols were correctly identified as

being negative, and th e 46 posi tive contro ls were identified as

conta ining oxazepam. The calibrators that containe d 125, 250,

500, or 1 000 ng/m L of nordiazepam, oxazepam, lorazepam,

temazep am, r and ~-hydroxy t r iazo lam

were al l correct ly identified with the exception of three 125-

n g / m L c a l i b r a t o r s i n w h i c h R E M E D i f a i l e d t o i d e n t i f y

temazep am as be ing p resen t . Al l manufac tu rer -supp l ied cal i-

brators and c ontrols were correct ly identified.

Urine spec imens used in th i s s tudy were f rom a varie ty o f

individuals representing such diverse groups as preemploy-

m en t applicants and pat ients of different age grou ps (neonates ,

adolescents , adults) be ing treated both as in- and ou t-pat ien ts

fo r a n a s s o r t m e n t o f s y m p t o m s , i n c l ud i n g t h o s e s y m p t o m s

requiring pat ients to at tend d rug rehabil itat ion programs. This

blend of pat ients wo uld be s imilar to those evaluated in oth er

medical centers th at u se EM IT immunoassays to analyze spec-

i m e n s a n d o b t a i n r e s u l t s t h a t a r e n o t n e e d e d i m m e d i a t e l y

because of cost considerat ions and avai labi l i ty of automated

ins t rume n ta t ion ; the T r iage methodo logy , on the o ther han d ,

is used mainly to analyze specimens and obtain results th at are

neede d imm ediately . These two screen ing technolo gies differ

because the an tibody used in the fo rmer techno logy i s gener-

a ted to uncon jugated oxazepam and an t ibod ies used in the

lat ter techn ology are genera ted to glucu ronide s of several d if-

ferent benzodiazepines. This difference is most notable for

detection o f lorazepam, which is excreted in urin e alm ost com-

pletely as the glucuronide; several s tudies (6 ,34) suggested

tha t EM IT wil l not dete ct lorazepam, whereas Triage wil l . Less

c lear f rom the l i te ra tu re was wheth er a s imi la r resu l t wou ld

also occu r with specimens c ontaining midazolam and i ts major

metabolites , ~-hydroxymidazolam and 4-hydroxymidazolam

(Table I and Figure 2). We studied this possibi l i ty by using

E M IT d . a . u , t o e v a l u a t e t h o s e s p e c i m e n s t h a t h a d b e e n

screened wi th e i ther EM IT o r T r iage and tha t subsequen t ly

h a d b e e n fo u n d t o c o n t a i n o n l y Io ra z e p a m o r m i d a z o l a m

metabo l i t es by REMEDi and GC-MS. Our resu l ts , un l ike tha t

o f o thers , sugges ted tha t lo razepam, w hen p resen t as the on ly

ur inary benzod iazep ine in amo un ts g rea ter than 1000 ng /mL,

will be dete cted by EM IT d.a.u. This preliminary finding will be

the sub jec t o f a subsequen t repor t. Also , we found tha t those

42



Journal of Analytical Toxicology, Vol. 20, October 1996

specimens containi ng cr as identified by

both REMEDi and GC-MS, were negative by EMIT d.a.u., and

this finding suggested that the benzodiazepine glucuronide-

derived antibodies in th e T riage device may be superio r for

detecting th e presence of midazolam metabolites. Additional

studie s will be required to validate this initial finding.

REMEDi analysis identified the presence of demoxepam in

360 specimens and suggested tha t the patients had used chlor-

diazepoxide because demoxepam can o nly be found as a result

of chlordiazepoxide metabolism (Figure 2). This finding was

not unexpected because a num ber of our specim ens were from

patients in drug rehabilitation programs that routinely use

chlordiazepoxide in withdrawal therapy. Clearly, REMEDi

analysis was advantageous in perm ittin g the deter mina tion of

chlordiazepoxide use in these patients because GC-MS analysis

could not determine the presence of demoxepam because of its

pyrolysis to nordiazepam, a common intermediate in the

metabolic pathway of many different 1,4-benzodiazepines

(Figure 2). Other benzodiazepines and th eir metabolites were

also found in combinatio n with dem oxepam and illustrate the

comm onality of pathway thr oug h nordiazepam as well as the

prescribing practice of using combination s of benzodiazepines

(e.g., chlordiazepoxide and lorazepam o r chlordiazepoxide and

alprazolam). Nordiazepam was found in less than 5 of the

specimens that also contained demoxepam, which suggests

that demoxepam is a principal urinary metabolite of chlor-

diazepoxide and therefore impo rtant in uri ne analyses of ben-

zodiazepines. The presence of oxazepam along with demox-

epam in the majority of these specimens also illustrates the

additional metabolic pathways for chlordiazepoxide, namely,

those passing through demoxepam, nordiazepam, and

0 . 2 5 ~

0 23 1

Z

o.17:

o 15;

0 . 1 3

~ 0 11

0 09

1 7

0.05

0.03

0.01

-0 .01

- 0 . 0 3

11

oxazepam. Figure 3 illustrates a typical HPLC chrom atog ram

from a patient's specim en in wh ich chlordiazepoxide and its

metabolites were identified.

Making an assignment of whether the patient has been

administered diazepam, clorazepate, or prazepam by analysis of

urine would be difficult because all three benzodiazepines pass

thro ugh the co mm on intermediate, nordiazepam. However,

only diazepam can give rise to temazepa m (Figure 2); there-

fore, identification o f temazepam along wi th nordiazepam and

oxazepam would sug gest diazepam administration. Clorazepate

is rapidly decarboxylated in the gastric jui ce of the gu t to no r-

diazepam (35); therefore, no unchanged drug would be

expected in the u rine. Furthe rmore, if ciorazepate is analyzed

by GC-MS, it is quantitatively converted to nordiazepam in th e

injector po rt env ironm ent (data not shown). Thus, the finding

of nordiazepam and oxazepam without t emazepa m suggests

tha t either clorazepate or prazepam was the administered drug.

Only one patient's specim en was found by REMEDi to contain

nordiazepam and oxazepam, as also confirmed by GC-MS, and

suggested that, in our population of patients, prazepam and

clorazepate were not widely used. B oth diazepam and prazepam

are excreted to a lim ited extent in urine, bu t REMEDi analysis

did not detect either of these compounds in the urine speci-

mens. Prazepam is known to be metabolized to 3-hydroxy-

prazepam, but this metabolite was not part of the REMEDi

library evaluated in our study. Likewise, the GC-MS analyses

used did not detect this metabolite or its parent drug or

diazepam. Further studies will be required to demonstrate

wheth er these additional metabolites and parent d rugs can be

identified in urine.

Based on our findings that less than 2 of all specimens

13

16

3 6

12 45

[ 1 12 14

I I I I i I I [ I i i I l I | I l I I I I i I J I I II l I I i I ~ I l l I ~ I I I I D i

1 2 3 5 6 7 8

I3me (min) 235 nm

analyzed by REMEDi or GC-MS conta ined

eit her cr or hydroxy-

ethylflurazepam, the prescribing of tria-

zolam and flurazepam appeared to be minor

in our study population. However, the use

of either of these drugs, as well as alpra-

zolam or clonazepam, was easy to differen-

tiate because each had unique and specific

metabolites detectable by both REMEDi and

GC-MS analyses.

Temazepam can be prescribed as a single

Figure 3. A high-performance liquid chromatographic chromatogram from a patient illustrating the

identification of chlordiazepoxide and its major metabolites in a urine specimen. In the chromatogram,

peak 9 (2.0 rain) is demoxepam, peak 11 (3.0 min) is oxazepam, peak 12 (3.6 m in) is chlordiazepoxide,

peak 13 (3.9 m in) is the internal standard triazolam, and pea k 16 (7.0 rain) is the internal standard ethyl

oxazepam (added as the glucuronide to the specimen).

drug entity, and, if this is the case, one

would expect to identify both temazepam

and oxazepam in the urine but not nor-

diazepam (Figure 2) because oxazepam is a

metabolite of temazepam. Evaluations of

the urine specimens in this study by

REMEDi demon strated th at six specim ens

met this criteria; two of these specimens

were also submitted to GC-MS and con-

firmed. Only one specimen was found th at

contained temazepam but not oxazepam

and that was observed with lorazepam also

present. Thus, these findings suggested tha t

when temazepam is administered as the

parent drug, both temazepam and oxaze-

pam will be found in the urine. Additional

4



Journal of Analytical Toxicology, Vo l. 20, O ctobe r 1996

s t u d ie s w i t h p a t i e n t s r e c e i v i n g o n l y t e m a z e p a m wo u l d b e

needed to verify this prel im inary finding.

A su rp r i s ing f ind ing in ou r s tudy was the p resence o f 7 -

aminoflunitrazepam detecte d by REM EDi and con firmed by

GC-MS in four spec imens . Al though a t the t im e o f the s tudy

we had reports tha t i l l ici t f luni traze pam was in our area, th is

was the fi rs t substant iat ion of these reports . This benzodi-

azepine is not approved for u se in the United States bu t has re-

portedly come into this cou ntry via S outh A merica and Mexico,

where i t is an approved drug.

Our evaluat ion of th is new REMED i methodology has sug-

gested tha t i t could have ut i l i ty in au gm entin g the identifica-

t ion of the parent benzodiazepine adminis tered to a pat ient

based on the urinary benzodiazepine metabolites found. This

was t rue in the case o f demoxe pam, which can ar i se on ly f rom

chlordiazepoxide adminis trat ion and cannot be identified by

GC-M S analysis . The m eth od also proved to be less compli-

cated w hen com pared with the arduou s procedures required to

p e r fo rm GC -M S a n a l y s e s . E v e n t h o u g h b o t h t e c h n i q u e s

requ i re an in i t i a l 2 -h hydro lys i s o f the g lucuron ides , the

rem aining instrumental par t of the analysis is weighed heavily

in favor of REM EDi because t he hydrolyzed urine, after a brief

centrifugation s tep, is p laced direct ly on the in strum ent, and

its analysis requires only abo ut 12 min before a resu l t can be

obtained. This assumes th at the i nstr um en t has been properly

cal ibrated for the day, w hich requires three additional sample

analyses, namely, Check Mix, Lim it Check, and a negative con-

trol. In contrast, GC -MS analysis requires approx imately an

additional 2 h of preanalytical preparat ion, includin g l iquid-

liquid extraction, ce ntrifugation, evaporation of the orga nic

solvent extract , derivat izat ion of mos t analytes , and an instru-

mental analysis t ime of 19-2 0 m in as well as solvent blanks

between e ach specimen t o preve nt carryover. Ag ain, as with

REMEDi analysis , GC-M S analysis requires prior in ject ion of

cal ibrators and controls . No nau tom ated H PLC analysis would,

l ike GC-M S, requ i re a p reanaly t ica l t rea tm en t o f the spec-

imen an d w ould also not offer the addit ional REM EDi advan-

tages o f UV spect ra l ma tch ing w i th a l ib rary and com puter

a lgor i thm fo r compo und iden t i f ica t ion based on two UVwave-

lengths and relat ive retent ion t imes using two internal s tan-

dards, one of w hich is a gluc uro nide to control hydrolysis .

GC-M S analysis had the advantage in sensi t iv i ty for detec-

t ion of the urine benzodiazepines and the ir metabolites . How -

ever, th is could be gained on ly by analyzing 3 m L of urine

instead of 1 mL an d by perfor m ing the analyses on relatively

new co lumns , wi th a new go ld -p la ted bo t tom in jec to r seal ,

and with a freshly cleaned ion source. W ithout these adjust-

me nts in the GC-M S methodology, we could no t obtain req-

uis i te sensi t iv i ty for most analytes , with the exceptions of

oxazepam and lorazepam, w ith th e 62.5-ng/mL in-ho use cal i-

brator. Thus, the sensit iv ity of the GC-M S meth od proved to be

h igh ly dependen t on the cond i t ion o f the ins t rumen t , and

a n a l y s e s r e p o r t e d h e re we re p e r fo rm e d o n l y u n d e r t h e s e

optimal parameters . This me ans th at typical wo rking condi-

t ions on a GC-M S ins t rum en t wou ld p rec lude m uch be t te r

sensit iv ity than the 80-ng/mL cu toff level claimed for REMEDi

by the m anufacturer. The qu est ion tha t was unanswered by this

s tudy was w hethe r a cutoff level of 80 ng/m L was adequate to

detec t the diazolo- and triazolobenzodiazepines in urine after

a therapeu tic dose, wh ich is typical ly only 5-10 of the dose

given for the 1 ,4-benzodiazepines. Because the majori ty of our

study was designed to analyze by REM EDi only those speci-

m ens th at we re posi tive by imm unoassay, som e false-negative

specimens may have resulted from patients on low therape utic

doses of the diazolo- and triazotobenzodiazepines. This was

reinforced by ou r finding of cr in one sup-

posed ly immun oassay-negat ive u r ine spec imen by REM EDi

and th i s sam e m etabo li te by GC-MS in a few spec imens tha t

we re n e g a t i v e b y b o t h R E M E Di a n d i m m u n o a s s a y . O t h e r

autho rs (6-8,36) reported th at thera peutic doses of alprazolam

may p roduce u r ine leve l s be low 80 ng /m L even thou gh one

repo rt claimed tha t EM IT was adequate to d etect alprazolam

use in pat ients receiving the dr ug (8).

In o ur opinion, the REMEDi methodology should prove to be

useful in laboratories t hat ro utinely perform high volu me s of

b e n z o d i a z e p i n e c o n f i rm a t i o n s o f i m m u n o a s s a y s c r e e n i n g

results . T he savings in t ime and labor cost as well as the addi-

t iona l in fo rmat ion ob ta ined concern ing the iden t i ty o f the

adminis tered drug wil l lead laboratory directors to use their

GC-MS ins t rumen ts fo r o ther ass ignments . Because o f the

large numbers o f fa l se-pos i t ive and fa l se-negat ive resu l t s

typical ly seen with the benzodiazepine immunoassay tests ,

REMEDi o r nonau tom ated HPLC or GC-MS analys is shou ld be

considered in cri t ical specimens in w hich the screen ing resul ts

are n ot con sis tent with cl inical or o ther invest igatory find-

i n g s . F u r t h e r , i f i t is n e c e s s a ry t o k n o w wh e t h e r c h l o r -

diazepoxide was the adm inis tered drug, th en REMEDi analysis

or an equivalent HPLC analysis should be considered.

c k n o w l e d g m e n t s

This work was f inanced , in par t , by g ran ts - in -a id f rom

BioRad Laboratories a nd fro m National In st i tu tes of Health ,

Pediatric Pharmacology Research Unit grant n um ber I U10 HD

31324-01. The au thors grateful ly acknowledge the assistance o f

Drs. Daniel Cashman, Louis Fink, and Alex Papas and th eir

respec t ive s ta f fs in supp ly ing u r ine spec imens f rom the i r

inst i tu t ion s for use in th is s tudy.

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1 6 : 6 7 - 7 1 ( 1 9 9 2 ) .

Manusc r ip t rece ived Apr i l 8 , 1996;

rev is ion rece ived Ma y 20, 1996.

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