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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.
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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.
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~-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 .
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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
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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;
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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
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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
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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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Manusc r ip t rece ived Apr i l 8 , 1996;
rev is ion rece ived Ma y 20, 1996.
4 2 4