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Veri cation of the Simultaneous Analysis of Heroin Addiction Treatment Compounds Using LC/MS/MS with a New Prelude SPLC
™
System
Overview
Purpose:
There are several compounds used for the treatment of heroin addiction.
These compounds include methadone, buprenorphine, norbuprenorphine, naloxone,
naltrexone , and their metabolites. The metabolites of interest are 2-ethylidine-1,5
dimethyl-3,3-diphenylpyrrolidine (methadone metabolite, aka EDDP), buprenorphine
glucuronide (buprenorphine metabolite), and norbuprenorphine glucuronide
(norbuprenorphine metabolite). All total, the analysis of these compounds for research
includes 8 analytes with 4 internal standards, that are commonly used in the treatment
of heroin addiction.
Methods:
Samples for this analysis were prepared in human urine. After the addition of
internal standard, they were injected for analysis using the
Thermo Scientific™
Prelude™ SPLC sample preparation
-liquid chromatography system. This system was
fitted with a Thermo Scientific™
Accucore
™ 100x3.0, 2.6 µm particle size column for
separation. Additionally, a Thermo Scientific™ TSQ Vantage™ mass spectrometer in
positive ion mode was used for analyte detection.
Results:
All 8 compounds were simultaneously verified using the Prelude SPLC
system and the TSQ Vantage MS. The resulting chromatography, correlation
coefficients, standard curve linearity, quality control data, and analyte transitions are
explained in the following sections to illustrate the success of this analysis.
Introduction
Several different compounds are currently used in the treatment of heroin addiction.
These compounds and their metabolites were analyzed for research using the new
Prelude SPLC system and a TSQ Vantage MS. This workflow takes advantage of a low
system volume to decrease solvent consumption and successfully quantify methadone,
2-ethylidine-1,5 dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine,
buprenorphine glucuronide, norbuprenorphine, norbuprenorphine glucuronide,
naloxone, and naltrexone. This work verifies a heroin treatment panel method
performed on the Prelude SPLC system. In order for this system to be evaluated, it
must fall within certain acceptance criteria. These set parameters are designed to
determine the success or failure of a particular LC-MS/MS workflow. These parameters
include, but are not limited to:
1) The lower limit of quantitation (LLOQ) and low quality control need to be
±
20% of
the expected concentration.
2) All of the remaining calibrators and controls need to be
±
15% in order for the
instrument to be successfully validated.
3) All of these requirements must be met for three consecutive days so that interday
and intraday accuracy and precision can be determined.
4) The signal in the blank following the highest standard may not exceed 20% of the
LLOQ signal. This factor is often called carryover.
Methods
Sample Preparation
Human urine was spiked with all 8 analytes and then serial diluted into a calibration
curve. Buprenorphine, norbuprenorphine, buprenorphine glucuronide, and
norbuprenorphine glucuronide had an analytical measurement range of 1.0 ng/mL to
100 ng/mL. Methadone, EDDP, naloxone, and naltrexone had an analytical
measurement range of 5.0 ng/mL to 500 ng/mL. Quality controls were also prepared in
human urine at three different levels. The urine aliquots were diluted with a combination
of water and methanol that contained internal standards. These samples are then
injected onto the system for analysis.
Liquid Chromatography
Chromatographic separations of all compounds were performed using Prelude SPLC
system, seen in Figure 1, equipped with an Accucore 100x3.0mm C18 analytical
column with 2.6 µm particle size. The system mobile phases consisted of 10mM
ammonium formate, 0.05% formic acid in water and 10mM ammonium formate, 0.05%
formic acid in methanol. The system needle washes were 60% water, 40% methanol,
and 0.5% formic (aqueous) and 45% isopropanol, 45% acetonitrile, and 10% acetone
(organic).
Mass Spectrometry and Data Analysis
The detector was a TSQ Vantage triple-stage quadrupole mass spectrometer with
HESI-II ionization probe in positive ion mode. Quantitation of results was performed
using Thermo Scientific™ LC
QUAN
™ software.
Results
Analyte result summa
Buprenorphine, norbupr
glucuronide were all pre
concentrations at 3.0, 4
naltrexone were prepar
concentrations of 15.0,
for each analyte. Metha
EDDP. Naloxone-d5 wa
Buprenorphine-d4 was
norbuprenorphine-d3 fo
transitions used for the
Compound
methadone
EDDP
naloxone
naltrexone
buprenorphine
norbuprenorphine
buprenorphine
glucuronide
norbuprenorphine
glucuronide
methadone-d9
naloxone-d5
buprenorphine-d4
norbuprenorphine-d3
FIGURE 1. Prelude SP
Table 1. Analyte Transi