Supplemental Materials
Cocaine/Cocaine Metabolite Assay Method
The LC-MSMS method used was adapted from our previously published method
for cocaine and BE (Lin et al., 2001). All reference material was purchased from
Cerilliant (Round Rock, TX). In brief, d3-deuterated cocaine, BE, EME, and norcocaine
were added to 1.0 mL aliquots of plasma as the internal standards. The pH of the plasma
was made acidic (≈ 4.0) by the addition of acetate buffer, and the mixture was extracted
using solid phase extraction (SPE). The eluant was evaporated and reconstituted with
methanol/0.1% formic acid in water mixture (10:90) and analyzed by LC-APCI-MSMS.
The mass spectrometer was operated in the selected reaction-monitoring mode.
Quadrupole Q1 was set to pass only the MH+ ions, which were caused to undergo
collision-induced dissociation in quadrupole Q2 to abundant product ions as follow:
cocaine and cocaine-d3 at m/z 304 to 182, and 307 to 185, respectively; BE and BE-d3 at
m/z 290 to 168 and 293 to 171, respectively; EME and EME-d3 at m/z 200 to 182 and
203 to 185, respectively; and norcocaine and norcocaine-d3 at m/z 290 to 168 and 293 to
171, respectively. The product ions were then monitored selectively by quadrupole Q3.
The concentrations of cocaine, BE, EME and norcocaine were determined from the peak
area ratios of the analyte divided by the peak area ratios of its internal standard and
comparison of the ratio with the calibration curve (2.5 to 750 ng/mL) that was generated
from the analysis of human plasma fortified with known concentrations of the analytes
and their internal standards.
Validation of the Cocaine/ Cocaine Metabolite Assay
Figure 1a shows a chromatogram of plasma fortified only with internal standards;
Figure 1b shows plasma fortified with analytes at the lower limit of quantitation (LLOQ)
of 2.5 ng/mL and internal standards. There was minimal background noise at the
retention time of the analytes (Figure 1a) and sufficient signal-to-noise at the LLOQ
(Figure 1b). Norcocaine and benzyolecgonine had the same transitions but eluted with
sufficient chromatographic separation.
The results of the method validation experiments are summarized in Table 1.
Specificity was determined from comparison of blank plasma fortified with internal
standard (6 different sources, N=3 per source) to aqueous samples fortified with internal
standard and analytes at the LLOQ (N=3). Peak areas at the transition and retention time
of the analytes were quantitated (often by forced integration) and the peak area ratios
compared to the mean peak area ratios of the LLOQ samples. As percent of LLOQ peak
area ratios, those for cocaine, EME and norcocaine were less than 5%; those for BE were
13% (Table 1), which is still below the 20% maximum considered optimal. Excellent
recovery was achieved with the SPE method. When peak areas of extracted samples
(N=5 per concentration) at 5, 50 and 650 ng/mL were compared to unextracted samples
(N=5 per concentration) at the same concentrations, mean recovery for all three
concentrations ranged from 80% for BE to 99% for EME. Similar recovery was achieved
for the internal standard that was tested at their concentration of use, 25 ng/mL (Table 1).
Precision and accuracy were determined at the LLOQ and the concentration of the three
quality control sample (QCs) concentrations, 5.0, 50 and 650 ng/mL. For intra-run
precision and accuracy, samples were run at an N=5; accuracy was within 12% of target
and precision measured as % coefficient of variance (CV) was within 7.4% (Table 1).
Inter-run precision and accuracy was determined from the mean results from five
analytical bathes which included the intra-run batch (N=5) and four others at N=3.
Accuracy was within 10% and precision within 9.5% (Table 1). To determine the
stability of processed samples, low and high QCs that had been used as part of the
precision and accuracy experiment were stored after their initial analysis either on the
autosampler (5 days, N=5 per concentration) or at -20˚C (7 days, N=3 per concentration)
and then reanalyzed along with freshly extracted calibrators and QCs. The mean result of
all analytes was within 13% of target (Table 1), demonstrating that processed samples
were stable for these periods of storage.
Figure 1. Chromatogram of a) blank plasma fortified with internal standards and b) blank
plasma fortified with analytes at the LLOQ and internal standards.
Table 1. Operating characteristics for LC-APCI-MSMS method for determination of
cocaine, BE, EME, and norcocaine.
Validation
Experiment
Analyte
Cocaine
BE
EME
Norcocaine
Specificity (mean peak area ratio in 6 blank plasmas fortified with internal standard as
%LLOQ)
2.59
13.0
4.99
3.37
Recovery (%, mean of 3 concentrations, deuterated internal standard at 25 ng/mL in
parentheses)
91.1 (93.1)
80.4 (81.6)
99.0 (98.2)
85.6 (89.3)
Intra-run precision and accuracy (% Target ± %CV, N = 5)
2.5 ng/mL
97.6 ± 2.9
94.0 ± 6.0
92.0 ± 4.8
111.2 ± 4.3
5.0 ng/mL
89.2 ± 2.0
100.0 ± 7.4
90.6 ± 2.6
88.4 ± 3.6
50 ng/mL
89.2 ± 1.6
91.2 ± 2.9
90.2 ± 2.4
90.0 ± 3.1
650 ng/mL
93.3 ± 4.1
93.1 ± 4.6
93.3 ± 4.4
91.9 ± 3.0
Intra-run precision and accuracy (% Target ± %CV, N = mean of 5 runs with 3-5
replicates/run)
2.5 ng/mL
96.8 ± 6.6
96.8 ± 7.4
95.2 ± 8.4
105.6 ± 9.5
5.0 ng/mL
90.4 ± 2.4
95.6 ± 4.6
92.6 ± 1.7
91.6 ± 2.2
50 ng/mL
93.6 ± 3.2
97.4 ± 4.5
95.4± 4.2
94.2 ± 3.2
650 ng/mL
96.6 ± 3.8
96.5 ± 4.4
97.8 ± 3.0
95.5 ± 2.2
Processed sample stability, 5 days on autosampler (% Target ± %CV, N = 5)
5.0 ng/mL
88.6 ± 6.8
102.0 ± 11.4
93.6 ± 2.8
87.0 ± 14.9
650 ng/mL
98.8 ± 10.0
94.5 ± 2.4
97.1 ± 2.2
92.6 ± 3.2
Processed sample stability, 7 days at -20˚C (% Target ± %CV, N = 3)
5.0 ng/mL
650 ng/mL
94.1 ± 1.5
97.8 ± 17.0
97.5 ± 2.1
106.1 ± 2.5
107.3 ± 2.2 103.2 ± 4.9 105.2 ± 2.4 106.9 ± 4.0