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Quantitative Confirmatory Analysis of the NIDA 5 Panel Using Prelude SPLC System and TSQ Quantum Ultra MS
Overview
Purpose
: Develop and validate a simple and efficient quantitative LC-MS/MS
method for SAMHSA-compliant confirmatory analysis of 5 panel drug using
novel HPLC system.
Methods
: Human urine containing the drugs were spiked with internal
standards, enzymatically hydrolyzed, and diluted.
Results
: The LC-MS/MS method was developed and validated to comply with
SAMHSA guidelines.
Introduction
Effective on October 2011, the new SAMHSA/NIDA guidelines allow
implementation of LC-MS technique to perform NIDA-5 panel, urine quantitative
confirmatory analysis. LC-MS/MS methods are often less complicated than the
previously implemented GC-MS/MS methods because they do not require
derivatization. The NIDA-5 panel requires 6 separate quantitative methods for
analysis of THCA, opiates, amphetamines, cocaine, phencyclidine and 6-MAM to
confirm immunomethod positive samples. Here we developed 6 methods using
a single sample preparation procedure, analytical column, mobile phase and
instrument configuration. The methods are implemented on new Thermo
Scientific™ dual channel Prelude ™ SPLC online sample preparation-liquid
chromatography system, which allows method execution in parallel with a
different method on each channel or the same method on both channels
multiplexed to a single mass spectrometer.
Serial MS detection of multiplexed methods improves mass spectrometer
utilization time, increases laboratory throughput and reduces analysis cost. The
syringe pumps and high-pressure, low-volume gradient mixing used in the
Prelude SPLC system provide enhanced LC performance including improved peak
shape and resolution, stable retention times and reduced solvent consumption.
Methods
Sample Preparation
The sample prep procedure includes glucuronide hydrolysis followed by dilution.
For each sample a 200-µL aliquot of urine was spiked with 10 µL of internal
standard solution and 100 µL of β-glucuronidase enzyme in ammonium acetate
buffer, pH=5.0. The samples were incubated at 60 °C for 2 hours. A 200-µL
aliquot of methanol was added to each sample to stop enzymatic reaction.
Samples were cooled down, centrifuged and diluted 20-fold with water, except
for THCA, which was diluted 2-fold with water. Then 20 µL of sample was
injected onto the LC-MS/MS system.
Liquid Chromatography
Chromatographic separations were performed with the Prelude SPLC system by
direct injection onto a Thermo Scientific™ Accucore™ PFP 50x2.1mm, 2.6 µm
analytical column. The column was maintained at room temperature. Mobile
phases A and B consisted of 10 mM ammonium formate with 0.1% formic acid in
water and methanol, respectively. Separate methods were set up to analyze 6-
MAM, BE, PCP, and THCA. One method was set up for the combination of
amphetamine, methamphetamine, MDA, MDEA and MDMA. A final method was
used for the opiates morphine and codeine along with hydromorphone,
hydrocodone, oxymorphone and oxycodone. Figure 1 shows the LC method for
analyzing the opiates.
Mass Spectrometry
MS/MS analysis was carried out on a Thermo Scientific™ Quantum Ultra™ triple
quadrupole mass spectrometer equipped with a heated electrospray ionization
(HESI-II) probe. MRM transitions for each compound are listed in Table 1.
Validation
The calibration standards and quality control (QC) samples were prepared by
spiking compounds into blank urine. Samples were processed as described in
the Sample Preparation section. Methods were validated in multiplexed mode.
Intra- and inter- method precision and accuracy were determined by analyzing a
calibration curve along with replicate QCs on three different days. Matrix effects
were determined by comparing peak area of samples processed in multiple lots
of urine to that of one process in water. Additionally for the opiates, we were
able to correlate results obtained with this method to those from a toxicology
laboratory validated method.
Data Analysis
Thermo Scientific™ TraceFinder™ software was used for data acquisition and
processing. Data were processed with ion ratio confirmation.
Results
For each method, performance wa
quantitation limits (LOQ) for some
demonstrate method capability. T
PCP and THCA; 5-2000 ng/mL for
10-2000 ng/mL for morphine, cod
(Figure 2). The intra-method preci
9.6%, <15.9% for PCP, BE, 6-MAM,
respectively. The inter-method pre
<7.0%, <15.3% for PCP, BE, 6-MA
respectively. These results are su
were seen and those were largely
The percent recovery for 8 spiked
80-120% (Table 3). Data collected
correlated well with toxicology lab
>0.99 (Figure 4). Implementation
with syringe pumps improved rete
shape and resolution, thus allowin
methods while still keeping good
FIGURE 1. LC method for separat
TABLE 1. List of NIDA 5 compou
LOQ and Linear range
Drug
MRM (Q: Quan
Amphetamine 136.1-91.3 (Q), 13
Methamphetamine 150.2-91.2 (Q), 15
MDA
180.2-135.2 (Q), 1
MDMA
194.1-163.1 (Q), 1
MDEA
208.1-163.1 (Q), 2
Benzoylecgonine 290.1-168.1 (Q), 2
THCA
354.3-336.3 (Q), 3
Phencyclidine 244.2 -159.1 (Q), 2
Morphine
286.11-152.
286.11-16
Codeine
300.2-152.1 (Q), 3
6-Acetylmorphine 328.1-165.1 (Q), 3