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Confirmatory Determination of Buprenorphine and Norbuprenorphine in Urine Using A High-Throughput LC-HRAM-MS/MS Forensic Methodology
M S
Overview
ass p
MS/MS
Purpose:
To demonstrate that the use of a two channel UHPLC
a
-
t
ith hi h l ti
t
(HRAM) MS i
d
Plus Hybr
sys em w a g reso u on accura e mass
equ ppe
heated el
with segmented quadrupole allowed for high specificity, sensitivity,
iti
i
and sample throughput
pos ve
.
settings f
Methods:
The LC-HRAM-MS/MS method was validated by injection
data Ta
f 8 i t lib ti
QC d d
l
i
t
d
.
t
o -po n ca ra on curves,
s, an onor samp es n s aggere
parame e
fashion across two LC channels and three days.
Results:
This methodology resulted in achieving a sample cycle time
of 1 07 minutes and dynamic linearity range of 5 – 2000ng/mL
.
.
Table 1.
I t d ti
n ro uc on
Forensic toxicology labs monitor levels of buprenorphine (BUP) and
Anal te
its major active metabolite, norbuprenorphine (NBUP), in urine for law
y
enforcement purposes LC-MS/MS is currently the most popular
.
method used for confirmation Here we demonstrate that the use of a
BUP
.
two-channel UHPLC system with a high resolution accurate mass
(HRAM) MS equipped with segmented quadrupole (Figure 1 – system
NBUP
view) allowed for highest specificity sensitivity and sample
,
,
th h t
d t t d d LC MS/MS
BUP-d4
roug pu as compare o s an ar -
.
NBUP-d
Methods
S l P ti
amp e repara on
A th ti
i
(S i
™ DYNA TEK I d t i
L
KS)
Table 2
syn e c ur ne ur ne ,
-
n us r es, enexa,
was
.
used for calibrator and QC preparations. Six drug-free donor urines
and an HPLC-grade water sample (in triplicate) were each prepared
t 100 / L d d f
t i
t d BUP NBUP th i d t
t d
HESI II
a
ng m an use or ma r x s u y.
,
, e r eu era e
analogs, BUP-d4 and NBUP-d3, and NBUP glucuronide (NBUP-
Sheath
Gluc) were purchased from Cerilliant Corp Round Rock TX A beta-
.,
, .
l
id (T L II f
P t ll
l t
Si
Ald i h C
Aux Ga
g ucuron ase ype - rom
a e a vu ga a,
gma- r c orp.,
St. Louis, MO) was used for hydrolysis of glucuronides. A sample
Sweep
containing the NBUP-Gluc was prepared at 500ng/mL in Surine
TM
to
S
monitor as a hydrolysis control Sample prep: To 200uL of sample
pray V
.
Capillar
(calibrant or QC) was added 100uL of the enzyme prepared in 1M
Acetate buffer pH4 and 10uL of an internal standard mix This was
Aux Ga
,
,
.
then hydrolyzed @ 60
0
C for 2 hours The reaction was stopped with
.
S-Lens
addition of 200uL of methanol and cooled with refrigeration for 10
*A bit
minutes Centrifugation was subsequently performed @ 10k rpm for
r rary
.
10 minutes and the supernatant diluted 10X with water LC MS/MS
. -
l i
f
d i h L i j
i
l
ana ys s was per orme w t 75-u n ect ons vo umes.
Table 3
FIGURE 1 T
d II LX 2 ith Q E ti
Pl
H b id
. ranscen - w xac ve us y r
Quadrupole-Orbitrap High-Throughput LC-MS/MS system.
Target
Polarit