Figure 4. Acid Hydrolysis of Benzodiazepines

7
R 9 4 |
N HCI ae
Se [as Tl i i
if [ ioe c: for 15 min AEN. ° |
ZN = — 7
lx YN x i x |
LA i > AR? |
! { a |
i
1
Figure 5. Enzyme Hydrolysis with 0- Giucurenidase

HAC ay 8

Nw 6 Ly |
ody a
_ OH

‘Yemazepam Glucuronide

N

—_-A _ lucuronidase
f i
“ INION=

_seomronpetaumsean pe mecemce te sapnnsmepeatin tats coscpe sigma apereemesesr meer



i
i
at

Pan
H

i

988 1989 1

his person was flying in violation of FAA

taken.

regulations, and our toxicological assessment aided in
the determination of accident causation.

Since the implementation af the new procedure
described in this paper, we have noted an increase in
the number of aviation fatality cases reported positive
for benzodiazepines (Fig. 9).

The new procedure includes:

© enzyme hydroly:

* New extraction methods

* Utilization of HPLC with photo diode array de-
tection

«Confirmation with negative chemical ionization

mass spectrometry (NCIMS)

Many of the positive benzodiazepine cases reported

in 1993 were found to contai cazolam and

temazepam. The relatively high number of posi

987, 1988, and 1989 were

escription

benzodiazepines found

most likely related co high

hal

er diazepam ¢

its in these years, as noted in Fig. 10.

igh dases as

Diazepam is prescribed in reiacively
compared to the newer potent benzodiazepines, such
as alprazolam and temazepam. This makes identifica-

tiow of the new benzodiazepines more difficul: than
the identification of diazepam. Prior to 1992
of benzod

diazepam or its mexabolite

no cases

u between

rote

the decreasing nu
cases being reported, and the dec

prescriptions for diazepam (figures 9 and 19).

though alprazolam has been heavily prescr

zolam was not being frequently derecced until

d 1993. when new CAMI anal

ical methods



ion Spectrum

of

Temezesem

Average of5.458 to SASS mln, rom temazepan.d

ua

ssiCharge

benzophenone during acid hydrolysis, as seen in (Fig.

4), Enzyme hydrolysis, on the other hand, preserves
the integrity of the original molecular structure of the
benzodiazepine. Enzyme hydrolysis, therfore, makes
it possible to identify the starting b
(Figs. 5.6)

urine over time. and for th's reason urine is often used

odiazepine
(8). Benzodiazepines accumulate in the

initially to scteen for benzodiazepines. After finding

benzodiazepines in urine one can analyze blood or
plasma to identify the specific benzodiazepine previ-

ously found in urine.

CAMI Methodology
We have developed a rugged and sensitive method
for the analysis of benzodiazevines using enzyme
hydrolysis followed by extraction of the anaiytes using
‘9 disc technology. Samples are confi

use of GC/MS and HPLC. A trimethyl

ed by<

jerivative

is used to improve the chromatographic characteris-

tics of benzodiazepines for the GC) e mobile

described |

phase and gradient program ogan (9}.

MATERIALS AND METHODS

Instrumentation

The HPLC was e Hew
equipped with photodiode array detecto
auto-injector linked to a Pascal Chemstation.

ORP s

column was an E. Merck-Lichrospher 60 RP select B,

acetonitrile and 0 mM P Phosphate buffer, pH 3.2

ga gradient elution with a flow of 1.3 ml/min.
ne HPLCoven temperature was 35°C. The injection
volume 10 pL monit 230 am, from 190

nm to 400 am,

ring signal



is of urine specim:

ograph after enzyme hydrotys:

S versus Rete:

Figure $. HPL

a
a

bance unt

Absor'

fy)

ae

caffeine

§
i]
2,
Y
N
w@
&
o
§ i
i
if
HM
5
ine
ts 2
§ Es
8 Sy
cy
E

cd

remetdee sen Ante pnttintnenenenntn

TET ETTT YT TT TT TTT en
10 41 42 13 44 45 M6 87 18 49 20 A 22 23 24 2S & 2t



© Mass Spectrometer was a Hewleu Packard

5989 MS Engine. The column was a crosslinked 5%

phenyl methyl Silicone of 15 meter in length, 0.25

fi

1 thickness.

ternal diameter, and 0.25

m was the carrier gas with a flow of 1.5 ml/min.
The injector, te!

temperatures were maintained at 250°C, 150-250°C,

program and transfer line

and 275°C, respectively. The acquisition was in elec-
tron impact mode scanning from 45 to 480 amu. The

injection volume was 1 pl in the splidess mode.

Sample Treatment

Five ml.s of urine is spiked with flurazepam as the

cernal standard, Two mLs of 0.1M Acetace Buffer
pH 5 is added along with 10,000 units b-Glucu-
vonidase to hydrolyze the specimen. The sample was
heated for 4 hours at 40 °C. {wo mls boric acid
solution and 100 ul of 11.8 N KOH are then ad
pH the sampie to 8.5 + 0.5.

Extraction
The Micro Disc was prepared by adding | m1. of

methanol to the column reservoir. This is followed by
the addition of 1 mi of Borate Buffer, pH 8.5. The
into the column reservoir and

sample was poured
aspirated through the disc. One ml. of H,O is added
and aspirated, through as a rinse. The vacuum on the
extraction box is increased to dry the disc for °
minutes. The analyte is cluted off with (CH,Cl,

isopropyl a.cohol (86:29) with 2% NH4OH] into a

conical cube.

Derivatization

The eluare was evaporated to dryness, using nit
gen. For analysis on the HPLC, 25 ph of metha
was added to the tube, vortexed and transferred to an
autosampler vial for injection onto the HPLC. For
Mass Spectrometer analysis, 50 pL of BSTFA with 19
YMCS is added to the evaporated eluate tube and

incubated for 30 minutes at 70°C. The tube is allowed

to cool and the contents is transferred to an autosam
vial. One pL is then injected onto the mass spectror
eter for confirmation.

Spectra of temazepam were acquired utilizin

electro! pact and negative chemical ioni
spectrometry (NCIMS) (Figs. 7 and 8).

ning tests using

tion immunoassay ofa urine specimen revealed 86 ng!
:

m! of ¢ benzodiazepine. Bleod from this case w

screened using radioimmunoassay; a benzo!

was detected at a level in excess of 260 a

m was detected in

presence of tem
ng HPT

No benzodiazepine was dezected in 1 the

Hine specimen, when it was initially

LC at an LOD of 16 ng/m
iden urine by the use of

‘ollowing enzyme

hydrolysis The
blood was found to contain 44 ng/ml o yepam,
83 ng/m! of fluoxetine, and 138 4 oF nor-

fluoxetine. All of these drugs were also confirmed in

was found to contain
145 nefg of temazepam but was not analyzed for

fluoxetine or norfluoxetine,
DISCUSSION AND CONCLUSIONS

Both the GC/MS and HPLC were demonstrated to

be adequate methods for the determination of a rep-

resentative benzodiazepine in biological specimens, at
subtherapeutic levels. Significant signal to noise ratio
above the baseline at the limits of detection (LOD) of
10 ng/mL of temazepam was observed. One can see c

mazepam peak in che post hydrolysis HPLC
chromaiogram (Fig. 6), wher

ompared to the same

urine specimen, which had previously been analyzed

without hydrolysis (Fig. 1).

In summary, although the pilor discussed in

this
report had been prescribed clonazepam, 2 drug used
for the treatment of seizures, the pilot was found to
actually be using temazepam, a sleep inducing agent.
The prescrigtion medication clonazepam was not
he findi

metabolite, norfluoxetine, confirmed the pilot was

found in this case

g of fluoxetine and its

taking one of the prescription medications found at

the accident. This pilot had not reported to the FAA,

as required, any of the drugs found in this case or the

medical conditions for which the drugs were being

—



DOT/PAA/AM-96/14

Office of Aviation Med
Washington, D.C. 20591

Final Report

ansportation
Fecerai Aviation
Administration



Figure 10. A Comparison of Diazepam anc Alprazolam Use Since 7

i
i
i
i

The Y axis represents ihe percentage of a particular benzociazepine
prescribed during the respective years based on the ranking of the top 203
prescriptions during that year.

Note: The 100 = most prescribed benzodiazepine

REFERENCES

Rall TW. Hypnotics and Sedatives. in: Goodman

and Gilmaan’s The Pharmacological Basis of Thera-

ipeutics, 8th ed. Gilman AG, Rall TW.Nies AS. ) . mpounds 3

"Yaylor P, Eds. Pergamon Press. NY 1999. 2 1 Toxicology. 1992

358.

Jones DW, Adams D, Mart . Rousseau R} é s, Division of Marion Laborato-
Diug Population in One ‘Thousand Geographi-

cally Distribuced Urine Specimens. Journal ofAna-

hytical Toxicology. 1985. 9:125-130

Fitzgerald RL, Rexin DA, Herold DA. Benzodiaz-

epine Analysis by Negative Chemical lon

Gas Chiomatography/ Mass Sj
nal of Analytical Toxicology. 1993.

nical Tor

Jones GR, Singer PP. The Newer Benzodiaze
In: Advances in Analy

Ed. Year Book Medical Publishers inc.
Locniskar A, Greenblatt DJ. O
Conjugative Biotransformatio
Biopharmaceutics and Drug Dispe
11:499-506

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Figure 2. Classical Benzodiazepine

nd

cl

Oxazepam

4
cy on ‘one . >? Gy _-O._pOOOr
I! oe: T ae

ANON i

Temazepam Giucuronide

zn!
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Figure 1. HPLC Chromatograph of Nen-hydrolyzed Urine Sampie
Absorbance units versus Retention Time in minutes

s

a

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THe ANALYSIS OF BENZODIAZEPINES

INTRODUCTION

Benzodiazepines are commonly prescribed for their
anxiolytic, sedative, hypnotic, anticonvulsant, and
muscle relaxant properties. Since the introduction of
chlordiazepoxide in 1961, more than 3000 com-
pounds have been synthesized and approximately 35
are in clinical use today (i). In 1985, benzodiazepines
were the second most identified drug found in urine
(2). Only nicotine was found more frequently than
benzodiazepines. Benzodiazepines were detected more
often then acetaminophen and there were almost
iwiceas many benzodiazepines as cannabinoids found
in urine

Demonstrating the presence of absence of a benzo-
diazepine in a biological sample can have important
legal and medical consequences. Identification of a
benzodiazepine in a driver's blood can help cozrobo-
rate an officer's statement of impairment. The identi-
fication of this drug in a person is necessary to ensure
compliance with existing laws and prescription orders
(3). The use of benodiazepines by pilots isa violation
of FAA regulations. Finding this drug in a pilot may

hi

with FAA regulations.

el

determine the cause ofan accident or compliance

Initial screening of a urine specimen collected from
the pilot of a fatal aviation accident indicated the
presence ofa benzodiazpine using fluorescence polar-
ization immunoassay (PIA). However, no benzodi-
azepines were detected in urine, when the urine was
extracted, without prior hydrolysis, and analyzed us-
ing high performance liquid chromatography (HPLC)
(Fig. i). The authors were convinced the specimen
did contain a benzodiazepine based on the initial
screening results using FPIA, and the facc that
temazepam had been identified by HPLC and con-
firmed by Mass Spectroscopy (MS) in blood. it was
postulated that the drug was not being detected be-
cause it was present in che form of a glucuronide
jugate. Our approach: detection of benzodi-
azepines in urine is presented.

IN FORENSIC

The Structure Activity

zre of the classical benzodiaze

“Phe struct:
) consists of a benzene
g. Electron with
the biological act
1 Fin che ortho position of che 5 pheny!
ring usually increases the potency and decreases the
half-life (e.g., diazepam, temazepam, and flurazepam).

Some of the newer benzodiazepines have involved
annealing of the 1,2 diazepine position with a triazolo

or imidazo ring (e.g., triazolam and alprazolam) (4).

Benzodiazepines are extensively metabolized in the
liver. Many of the metabolites are physiologically
active. Metabolism can result in N-Alkyl compound
formation. Glucuronide conjugates, which are usu-
ally inactive, are typically formed by hydroxylated
metabolites (1). Femazepam will biotransform, via
N-demethylation, co oxazepam and bozh temazepam
and oxazepam metabolize to their glucuronide conju-
gate (Fig. 3). Temazepam and oxazepasa are recovered

in the urine primarily as the glucuronide conjugates (5).

Analytical approaches
Urinary benzodiazepines as a group are difficult to

identify and quantitate for several reasons (6):

°They are extensively metabolized to the glucu-
ronide conjugate, making it difficuir co extract
and identify.

¢ Some of the newer benzodiazepines are more po-
tent, and are found in the urine at low
concentrations.

° The

matography (GC) analysis, unless they are

tend to be thermaily labile during gas
derivatized or converted to their benzophenone
form by acid hydrolysis.
Many assays incorporate acid hydrolysis to cleave
off the glucuronide for analysis (7); however, the

benzodiazepine structure is altered by conversion toa