Jul 13, 2007 - grade or better; all reagents were ACS grade and .... Information, descriptions, and specifications in this publication are subject to change.
Determination of Buprenorphine, Norbuprenorphine, and Their Glucuronides in Urine Using LC/MS/MS Application Forensics
Authors
Introduction
Christine Moore, Cynthia Coulter, and Katherine Crompton Immunalysis Corporation 829 Towne Center Drive Pomona, CA 91767 USA
Buprenorphine is a member of the opioid family of drugs for the treatment of chronic pain, and in heroin addiction, as an alternative to methadone. It is metabolized to norbuprenorphine, and both species undergo extensive conjugation with glucuronide before urine excretion. The simultaneous determination of buprenorphine, norbuprenorphine, and methadone has recently been published [1]. Liquid chromatographic methods for the detection of buprenorphine in urine have predominantly been directed towards the free drug following hydrolysis, centrifugation, and/or extraction [2–4]. However, in 2003, Kronstrand et al. were the first to report on the detection of both free and conjugated compounds in urine using LC/MS/MS, noting that a low concentration of 20 ng/mL of free compounds seemed appropriate for the testing of patients. They improved the detection limit by hydrolyzing the specimens and subjecting them to solid phase extraction [5].
For purposes of contact only: Michael Zumwalt Agilent Technologies, Inc. 9780 S. Meridian Blvd. Englewood, CO 80112 USA
Abstract A rapid, simple, highly sensitive procedure for the simultaneous analysis of buprenorphine, its metabolite norbuprenorphine, and their glucuronides in urine using the Agilent 6410 Triple Quadrupole Mass Spectrometer in electrospray mode is described. Sample preparation included dilution of the urine samples in deionized water for direct injection into the LC/MS/MS system. Since the glucuronides are monitored in the same assay as the free drugs, no hydrolysis or extraction was necessary. To our knowledge, the procedure is the first to include the simultaneous monitoring of a qualifying ion for the parent drug, which is required to be present within a specific ratio to the primary ion for acceptable identification (± 20%). The Agilent MassHunter software allows the transitions to be monitored and automatically calculated into ratios, which must fall within the range of the calibration standards in order to be considered positive. While monitoring a qualifying ion naturally inhibits the sensitivity of the assay, the additional confidence in the result is a critical factor in forensic analysis.
In this work, we present a rapid method, sensitive to 1 ng/mL of urine, for the detection of buprenorphine, norbuprenorphine, and their glucuronides in urine involving simple dilution of authentic urine samples with deionized water. Two transitions per compound are monitored for the free drugs and one transition for the glucuronides. The monitoring of the qualifying ion and calculation of its ratio to the intensity of the primary transition are integral parts of the software package and necessary for forensic identification. The method is simple, sensitive, and rapid, with all analytes being determined in less than 8 minutes.
Experimental
Analytical Procedure
Materials and Methods
Instrument:
Agilent 1200 Series RRLC; 6410 Triple Quadrupole Mass Spectrometer
Standards and Reagents LC Conditions:
D4-Buprenorphine (D4-BUP); D3-Norbuprenorphine (D3-NBUP); BUP; NBUP; BUP glucuronide; and NBUP glucuronide were purchased from Cerilliant (Round Rock, TX). All solvents were of HPLC grade or better; all reagents were ACS grade and purchased from Spectrum Chemical (Gardena, CA). Internal standard mix: D4-BUP; D3-NBUP (1,000 ng/mL) Unlabelled drugs: BUP, NBUP, BUP glucuronide, NBUP glucuronide Extraction Procedure–Urine To urine (0.1 mL), add deionized water (0.35 mL) and 0.1 mL internal standard (1 µg/mL)
Column:
ZORBAX Eclipse XDB C18 4.6 mm × 50 mm × 1.8 µm (PN: 822795-902)
Dimensions:
4.6 mm × 50 mm × 1.8 µm
Column temp:
40 °C
Injection volume:
5 µL
Solvent flow rate:
0.8 mL/min
Pump Program: Time (minutes)
% 20 mM ammonium formate (A)
0 2.5 5 8.5 10
40 40 0 0 40
Post time:
3 min
% Methanol (B) 60 60 100 100 60
Calibration Curve: a) Negative:
0.1 mL D4-BUP; D3- NBUP
Mass Spectrometer Conditions: Operation:
Electrospray (ESI) positive mode
b) 1 ng/mL:
0.1 mL D4-BUP and D3-NBUP 10 µL of BUP, NBUP, and their glucuronides (100 ng/mL)
Gas temperature:
300 °C
Gas flow (N2):
6 L/min
0.1 mL D4-BUP and D3-NBUP 5 µL of BUP, NBUP, and their glucuronides (1,000 ng/mL)
Nebulizer pressure:
50 psi
Capillary voltage:
4500 V
Dwell Time:
50 ms
c) 5 ng/mL:
d) 10 ng/mL:
0.1 mL D4-BUP and D3-NBUP 10 µL of BUP, NBUP, and their glucuronides (1,000 ng/mL)
e) 20 ng/mL:
0.1 mL D4-BUP and D3-NBUP 20 µL of BUP, NBUP, and their glucuronides (1,000 ng/mL)
f) 40 ng/mL:
0.1 mL D4-BUP and D3-NBUP 40 µL of BUP, NBUP, and their glucuronides (1,000 ng/mL)
g) 100 ng/mL:
0.1 mL D4-BUP and D3-NBUP 100 µL of BUP, NBUP, and their glucuronides (1,000 ng/mL)
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The MRM transition settings are shown in Table 1. The NBUP and BUP have both quant and qual (in parenthesis) product ions.
Table 1.
Buprenorphine Acquisition Parameters Precursor ion
Compound Group 1
Fragment ion
RT (min)
Fragment voltage (V)
CE (V)
D3-NBUP
417.4
399.3
1.16
240
40
NBUP 3 gluc
590.5
414.4
0.73
240
40
NBUP
414.4
340.4
1.17
240
35
(187.2)
1.17
240
40
Group 2 D4-BUP
472.5
400.4
6.62
240
45
BUP 3 gluc
644.5
468.4
5.21
240
40
BUP
468.4
414.4
6.68
240
35
(396.1)
6.68
240
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( ) Qualifier ratios must be within 20% of calibration point
LC/MS/MS Method Validation The analytical method was validated according to standard protocols, whereby the limit of quantitation, linearity range, correlation, and intra- and inter-day precision were determined via multiple replicates over a period of 4 days. The slope of the calibration curve was not forced through the origin. The equation of the calibration curves and correlation coefficients (R2) are shown in Table 2; the precision and accuracy of the assay are shown
in Table 3. The assay was robust, precise, and accurate at the selected point of 10 ng/mL and was linear over the range of 5 to 100 ng/mL. The precision for all drugs was less than 20% both within day and between days, with most showing a variation of less than 10%. The limit of quantitation was 5 ng/mL; the limit of detection was 1 ng/mL. Figure 1 shows a typical calibration curve for buprenorphine, with a correlation coefficient of 0.9984.
Buprenorphine R2 > 0.998 1 – 100 ng/mL in urine
Figure 1.
Calibration curve for free buprenorphine in urine.
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Table 2.
Linearity, Correlation Coefficient, and Acceptable Qualifier Ratio for Buprenorphine and Related Compounds in Urine
Drug Buprenorphine
Calibration equation Y = 0.0065x – 0.005
Correlation coefficient (R2) 0.9984
Acceptable qualifier ratio (20%) 35.4 (28.3–42.5)
Norbuprenorphine
Y = 0.0068x – 0.0036
0.9995
44.9 (35.9–53.9)
Buprenorphine 3 glucuronide
Y = 0.0226x – 0.0064
0.9927
Norbuprenorphine 3 glucuronide
Y = 0.0013x – 0.0039
0.9948
Table 3.
Inter-Day Precision (10 ng/mL Control Specimens; n = 10)
Drug
Mean recovery (ng/mL)
SD
Precision (%)
Buprenorphine
10.74
1.38
12.85
Norbuprenorphine
10.08
1.36
13.51
Buprenorphine glucuronide
12.68
2.41
19.02
Norbuprenorphine glucuronide
11.1
1.84
16.55
Drug
Mean recovery (ng/mL)
SD
Precision (%)
Buprenorphine
10.22
0.58
5.64
Norbuprenorphine
8.76
0.57
6.54
Intra-Day Precision (n = 5)
Buprenorphine glucuronide
10
0.8
7.04
Norbuprenorphine glucuronide
8.98
0.61
6.75
Discussion The instrumentation allowed the rapid determination of buprenorphine, norbuprenorphine, and their glucuronides at low concentration, as is required for these drugs. The chromatographic separation produced by the small particle analytical column allowed separation of the peaks in each group segment (Figure 2). The software provided with the instrument is able to monitor a secondary transition from the precursor ion and automatically calculate the ratio to the primary ion. If the ratio is not within 20% of a calibration standard,
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the identification is rejected. This is an additional feature of the triple quadrupole mass spectrometer, which is extremely important in forensic analysis where court challenges to laboratory data are frequent. Monitoring a second transition gives additional confidence in the result; applying a ratio to that second transition compared to the primary product ion is a further enhancement to the identification of drugs in blood. The software plots the ratio in the chromatographic window, so the operator is able to assess positivity visually using the “uncertainty” band imposed by the software (Figure 3).
Norbuprenorphine glucuronide
D3-Norbuprenorphine
Norbuprenorphine - quant
Norbuprenorphine - qual
Buprenorphine glucuronide
D4-Buprenorphine
Buprenorphine - qual
Buprenorphine - quant
Figure 2.
Buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide extracted from authentic urine specimen.
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Figure 3.
Free norbuprenorphine (98 ng/mL) in urine: quantitation ion at left and overlay of quantitation ion with qualifier ion at right.
Conclusions The procedure described is suitable for the detection of buprenorphine and norbuprenorphine glucuronides in urine, without need for hydrolysis or extraction using an Agilent Technologies 6410 Triple Quadrupole LC/MS/MS system. This is the first method, which includes qualifying ions required to be present within a specific ratio, for the identification of buprenorphine and norbuprenorphine at low concentration in urine. The method is in routine use in our laboratory.
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References 1. L. Mercolini, R. Mandriolo, M. Conti, C. Leonardi, G. Gerra, M. A. Raggi, “Simultaneous Determination of Methadone, Buprenorphine and Norbuprenorphine in Biological Fluids for Therapeutic Drug Monitoring Purposes,” J Chromatogr. B Analyt Technol Biomed Life Sci 847(2): 95–102 (2007) 2. A. Tracqui, P. Kintz, P. Mangin, “HPLC/MS Determination of Buprenorphine and Norbuprenorphine in Biological Fluids and Hair Samples,” J Forens Sci 42: 111–114 (1997) 3. E. I. Miller, H. J. Torrance, J. S. Oliver, “Validation of the Immunalysis Microplate ELISA for the Detection of Buprenorphine and its Metabolite Norbuprenorphine in Urine,” J Anal Toxicol 30(2): 115–119 (2006) 4. E. J. Fox, V. A. Tetlow, K. L. Allen, “Quantitative Analysis of Buprenorphine and Norbuprenorphine in Urine Using Liquid Chromatography Tandem Mass Spectrometry,” J Anal Toxicol 30(4): 238–244 (2006) 5. R. Kronstrand, T. G. Selden, M. Josefsson, “Analysis of Buprenorphine, Norbuprenorphine and Their Glucuronides in Urine by Liquid Chromatography-Mass Spectrometry,” J Anal Toxicol 27: 465–470 (2003)
For More Information For more information on our products and services, visit our Web site at www.agilent.com/chem. For more details concerning this note, please contact Michael Zumwalt at Agilent Technologies, Inc.
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Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. © Agilent Technologies, Inc. 2007 Printed in the USA July 13, 2007 5989-7072EN
