Poster demonstrating the results from development/verification project for the quantitation of buprenorphine and norbuprenorphine in human urine.

1. Measurement of Urinary Buprenorphine and Norbuprenorphine by Liquid Chromatography-Tandem Mass Spectrometry
D. M. Garby, R. DelRosso, L. A. Cheryk
Mayo Medical Laboratories, Andover, MA
Introduction
Buprenorphine is a semi-synthetic opioid derived from the
alkaloid of the opium poppy, Papaver somniferum.1
Buprenorphine acts as a partial antagonist of the opioid mu
receptor and as such it has been successfully used as
substitution therapy for opiate dependance.1,2 Although
buprenorphine can be abused, its maximal effects are much
less than that of full mu agonists such as heroin and
methadone.2 This in turn, enhances the safety profile of
buprenorphine, with it less likely to result in abuse as well as a
reduced potential for diversion.2 In addition, buprenorphine in
combination with naloxone (available as Suboxone®)
discourages patients dependent upon mu opioid receptor
agonists (i.e., heroin, methadone and oxycodone) from
injecting dissolved tablets which would result in a precipitated
withdrawal syndrome.2
Buprenorphine is metabolized by N-dealkylation to
norbuprenorphine primarily via cytochrome P450 (CYP)
3A4.3 Buprenorphine and norbuprenorphine then undergo
glucuronidation to their inactive conjugated metabolites.1
Norbuprenorphine once formed has one fiftieth the activity of
buprenorphine, and is believed to play a minimal role in the
overall effects of buprenorphine.1
Treatment with buprenorphine generally consists of sublingual
doses of 12 to16 mg per day to treat opiate dependance.2
Dosage is adjusted in 2 to 4 mg increments/decrements to
achieve a level that suppresses opioid withdrawal.2
Monitoring of urine buprenorphine levels allows clinicians to
evaluate patient compliance during therapy and to detect elicit
use.
Assay Principle
Deuterated stable isotopes (buprenorphine-d4 and
norbuprenorphine-d3) were added to 500 µL of human urine
as internal standards. Sodium acetate buffer (pH=5.0) and
Glusulase® (>90k units glucuronidase) were then added.
This mixture was incubated at 50ºC to liberate the
conjugates. Buprenorphine, norbuprenorphine and the
internal standards were then extracted from the urine sample
by liquid/liquid extraction using n-butyl chloride at a basic
pH. The organic layer was removed, dried under nitrogen
and reconstituted with a solution of deionized water and
methanol (95/5 v/v) containing 0.1% formic acid.
The mobile phases for analytical chromatography consisted
of an aqueous phase (deionized water, 0.1% formic acid) and
an organic phase (acetonitrile, 0.1% formic acid). The
Zorbax Eclipse XDB-C18 4.6 x 50 mm, 3.5µm (Agilent
Technologies, Santa Clara, California) was used as the
analytical column. The LC-method was multiplexed
utilizing a TLX-2 chromatography system (Thermo Fisher
Scientific, Waltham, Massachusetts).
Final analysis was performed using a tandem mass
spectrometer (API 5000, Applied Biosystems, Framingham,
Massachusetts) equipped with an electrospray ionization
source in positive mode. The following operating conditions
were utilized: source temperature of 600°C, nebulizer gas
(GS1) at 60 psi, heated gas (GS2) at 60 psi, and curtain gas at
35 psi. Ion transitions monitored in the multiple reaction
monitoring (MRM) mode were m/z 468.3 → m/z 414.3 and
m/z 468.3 → m/z 396.2 for buprenorphine and its ion pair,
respectively, m/z 414.2→ m/z 340.2 and m/z 414.2 → m/z
364.2 for norbuprenorphine and its ion pair respectively, m/z
472.3 → m/z 400.3 for buprenorphine-d4 and m/z 417.2 →
m/z 343.2 for norbuprenorphine-d3. Calibrators consisted of
7 standard solutions ranging from 0 to 100 ng/mL.
Reproducibility
Intra-assay and inter-assay precision was demonstrated using tri-
level control material made internally. Intra-assay precision was
determined from 20 serial measurements of each sample. Inter-
assay precision was calculated from 10 replicate determinations
of each sample. The mean, coefficient of variation (CV) and
standard deviation (SD) were calculated at each level for each
method.
Recovery & Linearity
Linearity was assessed using serial dilutions of normal human
urine spiked with buprenorphine and norbuprenorphine standard.
Each level was run in triplicate and plotted versus the expected
concentrations. Recovery was determined with a mixing study
using high and low value control material.
Method Comparison
The LC-MS/MS method was compared to an LC-MS/MS
method (N=40) performed by an external reference laboratory.
Reproducibility
Buprenorphine Intra-assay (n = 20) Buprenorphine Inter-assay (n = 10)
Mean ± SD
(ng/mL)
%CV Mean ± SD
(ng/mL)
%CV
0.8 ± 0.02 2.0 0.8 ± 0.03 3.9
13 ± 0.1 1.1 13 ± 0.4 3.3
40 ± 0.5 1.1 41 ± 1.7 4.2
Norbuprenorphine Intra-assay (n = 20) Norbuprenorphine Inter-assay (n = 10)
Mean ± SD
(ng/mL)
%CV Mean ± SD
(ng/mL)
%CV
0.8 ± 0.03 4.2 0.8 ± 0.07 8.9
14 ± 0.4 2.8 13 ± 0.4 2.9
43 ± 0.7 1.6 41 ± 1.9 4.7
Recovery
Buprenorphine Recovery Study
Expected
Buprenorphine
Concentration
(ng/mL)
Measured
Buprenorphine
Concentration
(ng/mL)
% Recovery
11 10 91
21 20 95
31 30 97
Norbuprenorphine Recovery Study
Expected
Norbuprenorphine
Concentration
(ng/mL)
Measured
Norbuprenorphine
Concentration
(ng/mL)
% Recovery
10 10 100
21 20 95
30 30 100
Method Comparison
Specimen Stability
Buprenorphine Effect of Storage
% change
Day 1
% change
Day 3
% change
Day 3
% change
Day 14
20 – 25 °C -1.5 -0.5 -7.0 2.3
2 – 8 °C -0.9 -5.8 -6.7 -0.6
– 20 °C -1.2 -3.0 -7.4 1.4
Effect of Multiple Freeze-Thaw Cycles
% change
1 cycle
% change
2 cycles
% change
3 cycles
% change
4 cycles
0.2 -1.4 -4.2 1.6
Norbuprenorphine Effect of Storage
% change
Day 1
% change
Day 3
% change
Day 7
% change
Day 15
20 – 25 °C -4.0 -2.1 -6.8 -0.2
2 – 8 °C -3.0 -12.8 -5.7 -8.9
– 20 °C -8.0 -9.7 -6.5 -0.4
Effect of Multiple Freeze-Thaw Cycles
% change
1 cycle
% change
2 cycles
% change
3 cycles
% change
4 cycles
-8.6 -6.8 -4.9 5.3
The new LC-MS/MS method demonstrates a high degree of correlation to the reference LC-
MS/MS method. The new LC-MS/MS method has the advantages of increased throughput
through multiplexing of the method and a lower limit of quantification compared to that offered
from the external reference method.
Interferences
The following substances were not found to affect the chromatography or integration of buprenorphine or norbuprenorphine when assessed at the
following levels:
Conclusions
We have developed a reliable assay for the analysis of
buprenorphine and norbuprenorphine in urine using liquid
chromatography-tandem mass spectrometry. The assay offers
acceptable low end sensitivity with excellent accuracy, precision
and throughput.
References
1. Elkader A, Sproule B. Buprenorphine. Clinical pharmacokinetics in the
treatment of opioid dependence. Clin Pharmacokinet 2005;44:661-80.
2. SAMHSA. Buprenorphine: a guide for nurses. (TAP 30)
http://buprenorphine.samhsa.gov/TAP_30_Certified.pdf
3. Trescot AM, Datta S, Lee M, Hansen H. Opioid pharmacology. Pain
Physician 2008;11:S133-53.
Results
3,4 – Methylenedioxyamphetamine (MDA) 375 ng/mL
3,4 – Methylenedioxymethamphetamine (MDMA) 375 ng/mL
3,4 – Methylenedioxy-N-ethylamphetamine (MDEA) 375 ng/mL
d-Amphetamine 750 ng/mL
d-Methamphetamine 750 ng/mL
Amobarbital 600 ng/mL
Butalbital 600 ng/mL
Pentobarbital 600 ng/mL
Phenobarbital 600 ng/mL
Secobarbital 600 ng/mL
alpha-Hydroxyalprazolam 500 ng/mL
Nordiazepam 500 ng/mL
Ion Suppression
The absence of ion suppression was noted when buprenorphine
and norbuprenorphine were infused as the method acquired data
for drug negative urine samples.
Specimen Stability
Specimen stability was assessed under ambient, refrigerated and
frozen storage conditions and the effect of multiple freeze-thaw
cycles was also evaluated.
Interference Studies
The potential for method interference from commonly
encountered drugs was assessed by spiking a known
concentration of buprenorphine and norbuprenorphine into a
multi-analyte quality control material, Bio-Rad Liquicheck
Toxicology Urine Control C4.
Ion Suppression
Ion suppression was assessed by infusing a constant
concentration of buprenorphine and norbuprenorphine into
patient urine samples devoid of buprenorphine,
norbuprenorphine or internal standards. An inverse peak in the
chromatography would be expected if ion suppression was
present.
Experimental Design
11-Nor-delta-THC-9-COOH 75 ng/mL
Benzolyecgonine 500 ng/mL
Ethanol 125 ng/mL
Lysergic Acid Diethylamide (LSD) 2.0 ng/mL
Methadone 500 ng/mL
Methaqualone 500 ng/mL
Codeine 2000 ng/mL
Morphine-3-beta-D-Glucuronide 2000 ng/mL
Phencyclidine (PCP) 50 ng/mL
Norpropoxyphene 500 ng/mL
Propoxyphene 500 ng/mL
Linearity
Buprenorphine Method Comparison
y = 0.9424x + 1.406
R2
= 0.9918
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Reference Laboratory LC-MS/MS
MayoLC-MS/MS
Norbuprenorphine Method Comparison
y = 0.9755x + 0.6519
R2
= 0.9875
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Reference Laboratory LC-MS/MS
MayoLC-MS/MS
Buprenorphine Linearity
y = 0.9917x- 0.1037
R
2
= 0.9996
0
20
40
60
80
100
0 20 40 60 80 100
Expected
Measured
Norbuprenorphine Linearity
y = 0.9941x - 0.0349
R2
= 0.9994
0
20
40
60
80
100
0 20 40 60 80 100
Expected
Measured