This document describes the validation of a method for determining 31 drugs of abuse in human urine using automated in-line solid phase extraction and liquid chromatography-mass spectrometry. Urine samples containing the drugs and internal standards were hydrolyzed to cleave conjugates. The samples were then extracted using mixed mode solid phase extraction cartridges in an automated system and analyzed in-line by LC-MS/MS. The method was validated over five runs according to FDA guidelines and found to be accurate (94.9-100.9% for most drugs) and precise (RSD mostly 5-10%). Representative chromatograms are shown and a sampling of validation results provided.

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Validation of a Method for the Determination of Thirty One Drugs of Abuse in Human
Urine Using Automated, In-Line, ITSP Solid Phase Extraction and Liquid
Chromatography Mass Spectrometric Detection
Thurman L. Allsup1
, Kenneth C. Lewis1,
Rick Youngblood2
and Kim Gamble2
1
OpAns, LLC, Durham NC, 27713, 2
MicroLiter Analytical Supplies, Inc., Suwannee GA, 30024
Abstract
A quantitative analytical method has been developed
and validated for the determination of 31 total (free
and conjugated) drugs used in pain management or
abuse in human urine after hydrolysis. Opiates,
benzodiazepines and their metabolites make up most
of the list of drugs and are two of the main drug
classes analyzed by pain management labs.
Historical analytical procedures use hydrolysis of the
urine to cleave the glucuronide conjugates followed
by the addition of reagents to prepare the hydrolysate
for extraction by SPE. Generally the extracted
sample eluant is evaporated to dryness in preparation
for derivatization to allow analysis by GC/MS. This
method is extremely labor intensive. The method
that has been developed here is fully automated to
quantitatively extract total drugs from urine using
mixed mode ITSP SPE devices and analyze the
samples in-line by LC-MS/MS. The sample
extraction and injection onto the LC-MS/MS were
both performed in-line by a PAL (CTC Analytics)
sample handling unit. Spiked urine samples with
stable label internal standards were hydrolyzed with
β-glucuronidase and without further preparation
applied to mixed mode ITSP devices for extraction.
The extracts were injected in-line onto an LC-
MS/MS using a Poroshell 120 (Agilent) column. The
extracted standard curve had a range of 50-
5000 ng/mL for all except 6-MAM and norfentanyl
which were 10-1000 ng/mL, and fentanyl which was
5-500 ng/mL. Selected reaction monitoring (SRM)
was used for quantitation. An additional SRM
transition was acquired for each compound. The ion
ratio of the quantitative transition to the confirmatory
transition was used in order to confirm the presence
of the drug.
This method has been validated for use in a CLIA
laboratory and has met FDA Bioanalytical Guideline
acceptance criteria. A summary of the results of this
validation is presented here and complete results are
available from MicroLiter.
Experimental
Sample Preparation
All standards and their stable label internal standards
were obtained from Cerilliant. Standards of the drugs
were prepared in urine at nominal concentrations of
50, 100, 250, 500, 1000, 2500 and 5000 ng/mL
(standards of 6-MAM and norfentanyl were 20% and
standards of fentanyl were 10% of these
concentrations). Stable label internal standards
(100 ng/mL of each) were added to the urine. β-
Glucuronidase (5000 Fishman Units) in 1 M pH 4.5
buffer was added to each 1 mL of urine sample and
heated at 60°C for 30 minutes.
ITSP SPE Method
ITSP Cartridges: UCT Styre Screen DBX 10mg
(Product No.: 07-UDBX10-20A)
A CTC Analytics PAL HTS sample handler was used
to prepare the samples. The PAL was configured
with a 100 µL syringe and two tray holders. Each
tray holder held 2 microplates, one of which was
designed to hold the ITSP hardware kit (Product No.:
07-ITSP-HW). The extraction protocol was as
follows:
Step Solvent
Volume
(L)
Flowrate
(L/sec)
Wash B 100 20
Wash A 100 20
Load Sample 200 5
Wash A 100 10
Elute1
B 100 5
Elute C 100 5
Mix 100 50
1 Samples are eluted into vials containing 200 µL of 100
mM ammonium acetate
Solvent A: Water
Solvent B: Tetrahydrofuran (THF): Methanol: Water:
Ammonium Hydroxide (NH4OH) (4:3:3:0.2)

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Solvent C: 5% NH4OH
Samples were analyzed in-line with the following
method:
Analysis Method
Instrument: Agilent 6430 triple quadrupole with
Agilent 1200 Rapid Resolution HPLC
Column: Agilent Zorbax Poroshell 120 EC-C18,
3x50mm, 2.7 m particles
Solvent A: Water with 0.1% Formic acid
Solvent B: Methanol with 0.1% Formic acid
Column Temp.: 30˚C
Flowrate: 0.8 mL/min
Gradient: 0.00 min (97% A), 0.1 min (97% A),
1.0 min (85% A), 3 min (80% A), 4.2
min (50% A), 7 min (45% A), 7.75 min
(0% A), 8.25 min (0% A), 8.3 min (97%
A),
Ionization Mode: Positive Ion Electrospray Ionization
MRM Conditions: Dynamic MRM Mode,
Drug Quant Confirm IS RT
(min)
Oxymorphone 302-284 302-227 305-287 1.03
Hydromorphone 286-185 286-157 289-185 1.29
Oxycodone 316-298 316-241 319-244 2.03
Hydrocodone 300-199 300-128 303-199 2.2
MDA 180-163 180-105 185-168 2.44
6 MAM 328-165 328-211 334-165 2.42
MDMA 194-163 194-105 199-165 2.55
O-Desmethyl
Tramadol
250-58 232.2 256-64 2.66
MDEA 208-163 208-105 213-163 3.07
7-Amino
Clonazepam
286-121 286-222 290-121 3.14
BZE 290-168 290-105 293-171 3.76
Norfentanyl 233-150 233-177 238-155 3.96
Tramadol 264-58 264-246 268-58 4.12
Cocaine 304-105 304-182 307-185 4.17
Methylphenidate 234-56 234-84 243-93 4.22
Tapentadol 222-107 222-121 225-107 4.33
Meperidine 248-174 248-220 252-224 4.39
Normeperidine 234-160 234-91 238-164 4.48
Fentanyl 337-188 337-105 342-105 4.74
Meprobamate 219-158 219-97 226-165 4.74
Midazolam 326-291 326-249 330-295 4.88
EDDP 278-234 278-249 281-234 4.91
Drug Quant Confirm IS
RT
(min)
Midazolam 326-291 326-249 330-295 4.88
EDDP 278-234 278-249 281-234 4.91
Alpha-Hydroxy
Midazalam 342-324 342-203 346-328 5
Buprenorphine 468-55 468-101 472-59 5.07
Nitrazepam 282-236 282-180 287-241 5.5
Propoxyphene 340-266 340-58 351-64 5.68
Flunitrazepam 314-268 314-239 321-275 5.73
Alpha-Hydroxy
Alprazolam 325-297 325-216 330-302 5.86
Methadone 310-265 310-105 319-105 5.88
Carisoprodol 261-176 261-97 268-183 6.14
Alprazolam 309-281 309-205 314-286 6.18
Results
The validation of this method consisted of 5 runs
over 5 days. Each run consisted of at least a standard
curve in duplicate and QC’s at 3 levels in
quintuplicate. Other parameters that were examined
include: LLOQ, recovery, specificity, and dilution
suitability.
Representative chromatograms of each class are
shown below. The remainders are available upon
request.
Oxycodone.

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MDMA
Cocaine
Tramadol
Methyphenidate
Meperidine
Fentanyl
Buprenorphine
Nitrazepam

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Propoxyphene
Methadone
Carisoprodol
A small sampling of the results of the five run validation is
provided below. The results provided are the averages of
the accuracy and precision across the five runs. The
complete validation results are available from MicroLiter.
Drug Accuracy Precision
Oxymorphone 94.9 6.74
Hydromorphone 93.0 6.76
Oxycodone 96.3 7.91
Hydrocodone 94.0 6.62
MDA 97.0 6.85
6 MAM 100.9 7.69
MDMA 98.7 5.78
Drug Accuracy Precision
O-Desmethyl
Tramadol
96.6 4.72
MDEA 98.1 5.68
7-Amino
Clonazepam
99.4 6.64
BZE 94.9 5.69
Norfentanyl 95.8 9.55
Tramadol 94.9 6.45
Methylphenidate 97.5 5.2
Cocaine 97.2 5.26
Tapentadol 97.2 5.64
Meperidine 98.1 5.72
Normeperidine
96.5 5.58
Fentanyl
98.1 6.77
EDDP
98.6 5.87
Midazolam
94.8 6.34
Meprobamate
96.9 6.00
Buprenorphine
98.6 6.20
Alpha-Hydroxy
Midazalam
96.1 6.94
Propoxyphene
94.1 6.16
Methadone
98.7 6.44
Nitrazepam
94.0 6.70
Flunitrazepam
95.6 6.69
Alpha-Hydroxy
Alprazolam
96.9 6.97
Carisoprodol
94.6 6.00
Alprazolam
94.7 6.56
The method is being revised to include the following
compounds: morphine, codeine, amphetamine,
methamphetamine, PCP, norbuprenorphine,
chlordiazepoxide, norpropoxyphene, flurazepam, 2-
hydroxyethyl flurazepam, prazepam, diazepam, 7-amino
flunitrazepam, clonazepam, oxazepam, alpha-hydroxy
triazolam, lorazepam, nordiazepam, and temazepam.