A Quantitative SLE LC TOF MS Method to Detect Multiple Drugs in Urine

A P P L I C AT I O N N O T E
Liquid Chromatography/
Mass Spectrometry
PerkinElmer, Inc.
Waltham, MA
A Quantitative SLE
LC/TOF MS Method to
Detect Multiple Drugs and
Drug Metabolites in Urine
Introduction
Liquid chromatography-mass spectrometry
(LC/MS) is commonly used to measure drugs
and their concentrations in extracts of
biological matrices. Triple quadrupole MS is
often employed for targeted quantitative
analysis, using multiple reaction monitoring
(MRM) mode. Here, the intensity of product ions resulting from the collisionally induced
fragmentation of a specified precursor ion is measured. Using MRM mode requires complex
method optimization for each compound and the measurement is limited to targeted
species. In contrast, time-of-flight (TOF) MS has higher mass resolution than quadrupole MS
and allows for post-acquisition data interrogation of drugs and metabolites not originally
specified in the experimental method. This latter process is not possible using triple
quadrupole MRM data, which only records information on those species originally specified.
This study was conducted to evaluate the performance of a quantitative LC/TOF MS
method to detect 52 drugs and drug metabolites in human urine. Drug standards in
urine were treated with glucuronidase and then prepared using supported liquid
extraction (SLE) prior to LC/TOF MS analysis. Heavy isotope labeled internal standards
were utilized to normalize for variations in extraction efficiency and instrument response.
Experimental
Table 1. Liquid Chromatography Parameters.
Flexar System: FX-15 UHPLC pump, autosampler, and column oven
Mobile phase A
Water containing 0.1% Formic acid
Mobile phase B
Acetonitrile containing 0.1% Formic acid
Autosampler needle wash
Acetonitrile (2 x 1mL post-injection flushes)
Sample injection
10 µL fixed loop, 8 injections
Flow rate
0.4 mL/min
Column temperature
45 °C
Brownlee™ SPP Phenyl-Hexyl column
2.1 x 100 mm, 2.7 µm, p/n N9308485
Pre-column filter
0.5 µm porosity stainless steel
Table 2. Liquid Chromatography Gradient.
Time (min)
%B
0
7
0.8
7
4.8
60
4.9
100
5.9
100
6.0
7
3 min equilibration at 7% B
Table 3. Mass Spectrometry.
Instrument and Software Parameters
PerkinElmer AxION® 2 TOF mass spectrometer
Chromera® software
Ultraspray™ 2 Dual Probe electrospray source
Positive pulse mode
3 spectra per second acquisition rate
m/z range 70 to 1250
Drying gas flow: 14 liters per minute at 350 °C
Endplate heater: Medium
Lockmass mode: 127.0727, 609.2807, Search span 50 mmu
Lockmass solution: methanol containing 0.1%
Lockmass calibrant concentration: 6 µg/mL each
Left ESI probe (lockmass): 20 psi, right probe (column): 80 psi
Capillary exit: 90 V, Skimmer: 25 V
Extracted ion chromatogram (EIC) tolerance: ± 0.03 u
Table 4. Reagents.
Reagent
Certified reference material (CRM)
drug standards
Ethanol- and drug-free human urine
(OH2040)
LC/MS grade water (LC365)
LC/MS grade acetonitrile (LC015)
2
Supplier
Cerilliant® (Round Rock, TX)
Golden West Biologicals® (Temecula, CA)
Honeywell Burdick & Jackson®
(Muskegon, MI)
Honeywell Burdick & Jackson®
(Muskegon, MI)
LC/MS grade methanol (A 456)
Fisher Scientific™ (Pittsburgh, PA)
LC/MS grade formic acid (A 117)
Fisher Scientific™ (Pittsburgh, PA)
Ethyl acetate (E196SK)
Fisher Scientific™ (Pittsburgh, PA)
Ammonium hydroxide (28%, 338818)
Sigma-Aldrich® (St. Louis, MO).
Glacial acetic acid (695084)
Sigma-Aldrich® (St. Louis, MO).
5 M ammonium acetate (09691)
Sigma-Aldrich® (St. Louis, MO).
β-glucuronidase (Patella vulgata, G 8132)
Sigma-Aldrich® (St. Louis, MO).
Melamine (lockmass low, M 2659)
Sigma-Aldrich® (St. Louis, MO).
Reserpine (lockmass high, R 0875)
Sigma-Aldrich® (St. Louis, MO).
Mass spectrometry. The lockmass solution was delivered from
the onboard pressurized calibration vials through an 8 cm length
of 250 µm ID PEEK tubing to a 0.5 µm porosity stainless steel
in-line filter (IDEX, Oak Harbor, WA). Solvent flow was regulated
by connecting a 110 cm length of 125 µm PEEK tubing from the
filter to provide backpressure. This setup yielded a flow rate
of 30 µL/min. The monoisotopic peak was used to generate all
EICs except for the di-halogenated compounds lorazepam-D4
and phenazepam-D4. The EICs for these two compounds were
generated using the A+2 peaks to avoid interference from
higher isotope peaks of their corresponding unlabeled analytes.
Sample preparation. ISOLUTE 400 µL 96-well SLE+ and sample
collection plates were obtained from Biotage® (Uppsala, Sweden).
Isotope labeled internal standards solution in methanol (0.4 mL
of 1 µg/mL for each drug except flurazepam) were added to
glass vials and dried for 45 min. at room temperature in a
Thermo Savant™ SPD2010 SpeedVac system. A concentrated
stock (2,000 ng/mL) of 52 analytes was prepared in drug-free
urine (Lot #G011704063) using Cerilliant® CRM neat solution
stocks. The concentrated stock was spiked into drug-free urine
aliquots to produce the final concentration levels, calibrators:
1, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 ng/mL (Lot
#G011704063), test samples: 5, 10, 25, 100, 250, and 750 ng/mL
(Lot #G011611003). 2 mL aliquots of spiked urine were added
to vials containing the dried internal standards to yield a final
internal standard concentration of 200 ng/mL for all samples.
20 µL of 5 M ammonium acetate, pH 5 and 30 µL of β-glucuronidase
(333.3 units/µL) were added to each sample, mixed, and incubated
at 40 °C for 30 min. Samples were returned to room temperature,
then 20 µL of concentrated ammonium hydroxide was added to
each vial and mixed. Four 400 µL aliquots of each sample were
added to four consecutive wells of the SLE+ plate on a Supelco®
vacuum manifold and a brief pulse of vacuum was applied to load
the samples. The samples were incubated for five min. at room
temperature and then eluted with two additions of ethyl acetate
(600 µL each) which were allowed to flow by gravity, followed by
a brief pulse of vacuum. Eluates were dried under a stream
of nitrogen at 40 °C for 40 min. with a microplate evaporator
(Evaporex® EVX-192, Covina, CA). Each sample well was reconstituted
with 350 µL of 5% acetonitrile in water containing 0.1% formic
acid and shaken for 10 min. at room temperature at 400 rpm on
an incubating microplate shaker (VWR, Radnor, PA). The four wells
for each sample were pooled by transferring to a single well of a 2 ml
96-well plate (1.4 mL total volume each) for LC-TOF-MS analysis.
Data analysis. Instrument response was expressed as analyte/
IS ratio using the heavy isotope labeled internal standard for
each analyte, with the exception of flurazepam which used
buprenorphine-D4 as a surrogate IS. Calibration curves were
generated from the 10 level calibration samples using a quadratic
fit with no weighting and unconstrained y-intercept. The upper
and lower concentration level of the curves was determined as
the range which produced a uniform distribution of residuals
at each included level and had an analyte/IS ratio imprecision
of ≤ 25 %CV. The resulting calibration curve parameters were
used to determine concentration values of the test samples.
Percent recovery of test samples was calculated as (average
measured concentration / spiked concentration) × 100.
Imprecision of test samples (%CV) was calculated as (standard
deviation in concentration units / average measured concentration)
× 100. The limit of quantitation (LOQ) was determined as the
lowest test sample concentration with imprecision of ≤ 25 %CV,
recovery between 75% and 125%, and a minimum of seven
replicate measurements. Limit of detection (LOD) was estimated
as: t99(n – 1) × s LOQ where t99(n – 1) is the one-tailed t-statistic for
n – 1 observations at the 99% confidence level and sLOQ is the
standard deviation at the LOQ in concentration units. The t99(n – 1)
value is 2.998 for eight replicates and 3.143 for seven replicates.
Results
Figure 1 shows an example LC/TOF MS analysis, the first replicate
for the 250 ng/mL calibrator level, with extracted ion chromatograms
for all of the drugs and internal standards. The complete dataset
was processed to generate calibration curves for each drug or
metabolite. The median r2 value for all calibration curves was
0.9937 (range 0.9588 to 0.9986). Two example calibration curves,
for flunitrazepam and morphine, are shown in Figure 2. Calibration
curves were applied to test samples to determine concentration
levels and the resulting data is summarized in Table 5. Recovery
values for all analytes at all levels averaged 87.1% (range 66.4
to 121.1%) and imprecision averaged 10.4 %CV (range 2.5 to
23.7 %CV). Limits of detection (LOD) were from 0.9 to 122.7
ng/ml and limits of quantitation (LOQ) ranged from 5 to 250 ng/
ml. The LOD and LOQ were not determined for sufentanil due to
recoveries below 75%. Heroin was not detected with this extraction
method, however, the heroin metabolite 6-acetylmorphine was
detected with an LOQ of 10 ng/mL. LOQs for meprobamate (100
ng/mL), α-hydroxyalprazolam (100 ng/mL) and benzoylecgonine
(250 ng/mL) were relatively high with this method and may be
improved with changes to the SLE+ extraction protocol.
Conclusions
The LC/TOF MS method outlined here is capable of simultaneous
quantitative measurement of a large number of drugs extracted
from human urine. The sample preparation method can readily
be automated to streamline laboratory workflows. Modification
of the sample preparation protocol may improve detection limits
of selected analytes.
Figure 1. Example extracted ion chromatogram of the 250 ng/ml calibrator sample.
Flunitrazepam
Flunitrazepam
0.50.5
Morphine
Morphine
R² R²
= 0.9959
= 0.9959
0.40.4
Analyte/IS Ratio
Analyte/IS Ratio
Analyte/IS Ratio
Analyte/IS Ratio
0.40.4
0.30.3
0.30.3
0.20.2
0.20.2
0.10.1
0.10.1
0.00.0
0 0
R² R²
= 0.9898
= 0.9898
5050
ng/mL
ng/mL
100
100
0.00.0
0 0
2020
4040
ng/mL
ng/mL
6060
Figure 2. Examples of analyte calibration curves.
3
Table 5. Summary of Method Performance Parameters.
Calibration
Curve
Analyte
Lower
Benzodiazepines
% Recovery
Upper
r2
LOD
LOQ
5
10
25
100
81.1
103.1
105.3
88.6
85.1
81.0
86.9
92.3
105.3
87.9
86.6
83.2
96.4
106.8
92.0
92.7
78.5
92.2
92.1
105.5
84.1
91.5
93.1
94.2
93.4
84.3
85.4
86.0
100.9
83.2
121.1
84.4
103.4
98.8
97.5
84.7
84.7
80.9
88.9
83.3
98.2
73.7
93.2
103.2
83.0
79.5
75.6
81.0
79.4
76.7
86.6
75.2
78.7
82.3
77.3
97.3
77.0
79.1
91.0
81.9
84.2
84.6
80.7
96.3
83.3
84.1
77.7
75.8
82.0
86.3
76.1
82.6
78.6
75.5
68.7
72.8
71.9
83.9
97.1
72.8
74.6
82.0
76.4
81.3
85.5
88.9
78.1
97.5
86.1
75.2
74.2
81.3
80.3
77.5
97.2
72.7
66.4
77.9
80.2
80.6
87.2
91.6
80.8
92.9
89.5
80.2
78.6
86.5
89.0
80.3
94.7
79.4
73.9
85.6
91.5
88.7
101.0
7-Aminoclonazepam
Alprazolam
Clonazepam
Diazepam
Flunitrazepam
Flurazepam
Lorazepam
Midazolam
Nitrazepam
Nordiazepam
Oxazepam
5
1
2.5
1
1
1
1
1
1
2.5
1
100
25
50
50
100
250
250
25
50
100
100
0.9930
0.9697
0.9952
0.9953
0.9959
0.9986
0.9945
0.9765
0.9908
0.9961
0.9928
4.0
2.6
2.4
1.8
1.0
1.0
2.8
2.2
3.6
1.5
2.3
10
5
5
5
5
5
5
5
5
5
10
Phenazepam
Temazepam
α-Hydroxyalprazolam
1
1
5
100
100
100
0.9944
0.9903
0.9908
1.9
2.4
39.4
5
5
100
2.5
1
10
1
1
1
1
1
nd
1
2.5
1
1
2.5
5
1
1
1
1
2.5
1
2.5
1
1
1
50
50
250
50
50
25
50
50
nd
50
50
25
25
50
25
50
50
100
50
100
50
25
50
50
50
0.9932
0.9982
0.9767
0.9950
0.9936
0.9735
0.9983
0.9985
nd
0.9962
0.9835
0.9917
0.9865
0.9898
0.9794
0.9930
0.9945
0.9984
0.9951
0.9945
0.9904
0.9668
0.9962
0.9977
0.9985
2.4
2.1
4.5
1.3
2.0
2.2
1.6
1.0
nd
1.3
1.9
1.6
1.2
1.7
2.4
2.0
1.2
2.7
2.2
1.9
2.0
2.2
4.7
1.6
10
10
10
5
5
5
5
5
nd
5
5
5
5
5
10
5
5
25
10
10
5
5
25
5
5
100
1
10
1
1
5
2.5
250
1000
25
50
50
50
100
100
0.9755
0.9753
0.9941
0.9719
0.9941
0.9895
0.9981
0.9900
3.3
122.7
2.7
4.1
2.2
2.4
3.5
3.2
5
250
10
10
5
10
10
10
10
5
1
50
2.5
250
100
25
1000
25
0.9588
0.9986
0.9955
0.9963
0.9937
6.9
0.9
1.2
19.1
1.6
10
5
5
100
10
% CV
250
750
97.9
100.0
98.8
101.1
5
10
25
100
16.4
15.7
8.3
8.4
7.7
6.6
17.0
9.3
21.0
7.6
8.7
9.8
11.9
5.5
6.8
5.5
9.3
9.8
5.6
11.4
8.7
6.8
11.4
18.5
17.4
14.4
8.1
7.8
18.2
17.5
19.6
11.7
15.2
18.7
8.8
10.5
8.3
12.4
14.0
8.1
7.1
15.8
11.1
17.6
18.0
13.6
9.0
9.4
9.1
18.3
8.8
12.3
9.1
11.9
8.9
6.7
4.5
18.8
4.5
8.6
7.7
3.7
2.5
10.9
17.0
13.2
9.5
14.5
13.7
16.9
10.2
11.7
5.8
18.9
15.1
14.6
9.3
15.7
13.4
10.9
7.4
10.1
8.4
8.8
8.2
9.3
7.8
11.0
9.2
7.7
12.8
8.1
9.1
14.5
11.4
7.5
8.9
3.6
5.6
8.0
5.3
10.3
6.8
4.6
6.8
15.7
4.8
6.4
7.8
3.7
4.7
23.7
12.7
11.8
9.3
11.5
14.8
4.6
9.2
13.3
12.6
2.5
8.7
9.4
11.0
6.0
15.9
6.7
10.0
22.1
9.5
8.0
18.8
2.9
2.8
7.3
7.2
3.5
3.6
10.8
10.3
250
750
n <8
4.5
3.9
5.4
9.0
Opioids
6-Acetylmorphine
Acetyl fentanyl
Acetyl norfentanyl
Buprenorphine
Codeine
Dihydrocodeine
EDDP
Fentanyl
Heroin
Hydrocodone
Hydromorphone
Meperidine
Methadone
Morphine
Naloxone
Naltrexone
Norbuprenorphine
Norfentanyl
Normeperidine
Norpropoxyphene
Oxycodone
Oxymorphone
Propoxyphene
Sufentanil
Tramadol
67.5
9.2
85.3
95.8
96.5
93.3
12.4
7
10.2
5
5
3.9
13.5
7
7
Stimulants
Amphetamine
Benzoylecgonine
Cocaine
MDA
MDEA
MDMA
Methamphetamine
Phentermine
72.9
78.5
93.5
89.6
87.3
88.8
83.4
80.4
102.0
94.3
86.0
93.2
81.9
94.2
78.4
103.9
77.1
77.6
109.8
76.1
83.8
68.9
75.1
81.0
77.7
71.4
73.6
108.5
96.9
95.5
11.3
103.0
18.5
16.9
15.7
109.2
99.9
11.1
17.1
15.1
5.0
13.3
Others
Carisoprodol
Cyclobenzaprine
Dextromethorphan
Meprobamate
Phencyclidine
110.0
95.5
96.8
103.4
97.2
107.0
S amples with fewer than 8 replicates are indicated in bold with the number of replicates listed in the last column (n<8)
nd = not detected
For research use only. Not intended for diagnostic procedures.
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16.5
3.1
9.4
6.1
9.5
5.1