Pain Management and Drugs of Abuse - Aquilant … Drugs of Abuse ... Alpha-Hydroxy Midazalam 342-324...

ITSP™

Pain Management Drugs

and Drugs of Abuse

Automated Sample Prep

The SPE sample prep methods described in these

application notes were developed using ITSP on CTC PAL

autosamplers to take full advantage of sample prep

automation. Benefits accrued include:

Improved chain of custody

Ease of inserting STAT samples in mid-run of assay

Reduced risk of carryover

Improved lab tech safety and productivity

Increased quality of results

Improved utility of capital equipment

Reduced costs of consumables, solvent, disposal

LC-MS/MS

Many labs are moving to LC-MS/MS in order to perform more

tests at lower cost in less time. LC-MS/MS offers a number

of advantages versus the traditional analytical techniques of

LC, GC, or GC-MS, including:

Wider compound coverage

Better sensitivity

Better specificity

Less sample prep

Drugs of Abuse Detection and Quantitation

The assays presented herein are to detect and quantitate

over 50 different pain management drugs and drugs of abuse

in human urine using ITSP SPE for automated sample prep

on-line with LC-MS/MS for analysis. The first assay is for

basic analytes, the second is for acidic analytes.

888-232-7840 • 770-932-6565

www.microliter.com

PO Box 808, Suwanee, GA 30024

http://www.microliter.com/

MicroLiter Analytical Supplies, Inc. ITSP - Pain Management Drugs and Drugs of Abuse Document # MLDC00013

www.microliter.com Page 1 Rev Date: 101220

Determination of Fifty One Pain Management Drugs and 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. Lewis

1, Rick Youngblood

2 and Kim Gamble

2

1OpAns, LLC, Durham NC, 27713,

2MicroLiter Analytical Supplies, Inc., Suwannee GA, 30024

Abstract

A quantitative analytical method has been developed

for the determination of 51 drugs (total, free and

conjugated) tested for in pain management or drugs of

abuse assays of human urine. 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

cartridges 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)

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 cartridges 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 10-5000 ng/mL for all

except for normeperidine, buprenorphine, and

norbuprenorphine which were 1-500 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.

Experimental

Sample Preparation

All standards and their stable label internal standards

were obtained from Cerilliant or Toronto Research

Chemicals. Standards of the drugs were prepared in

urine at nominal concentrations of 10, 20, 100, 200, 1000,

2000 and 5000 ng/mL. Stable label internal standards

(50 ng/mL of each) were added to the urine. β-

Glucuronidase (10000 Fishman Units) 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

( µL)

Flowrate

( µL/sec)

Load Sample 500 5

Wash A 100 5

Elute B 100 5

Elute C 100 5

Dilute A 200 50

Mix 100 50

Solvent A: Water

Solvent B: Tetrahydrofuran (THF): Methanol: Water:

Ammonium Hydroxide (NH4OH) (4:3:3:0.2)

Solvent C: 5% NH4OH



Samples were analyzed in-line with the following

method:

Analysis Method

Instrument: Agilent 6410 triple quadrupole with Agilent

1200 Rapid Resolution HPLC

Column: Agilent Zorbax Poroshell 120 EC-C18, 3x50mm,

2.7 µm particles

Solvent A: Water with 0.1% Formic acid

Solvent B: Methanol with 0.1% Formic acid

Column Temp.: 50˚C

Flowrate: 0.8 mL/min

Gradient: 0.00 min (97% A), 0.1 min (97% A), 1.0 min

(75% A), 1.2 min (55% A), 3 min (40% A), 3.2

min (40% A), 3.25 min (5% A), 3.75 min (5% A),

3.8 min (97% A),

Ionization Mode: Positive Ion Electrospray Ionization

MRM Conditions: Dynamic MRM Mode, Δ RT 0.3 min for all

drugs

Drug Quant Confirm IS

RT

(min)

Morphine 286-165 286-201 289-165 0.83

Oxymorphone 302-284 302-227 305-287 0.89

Hydromorphone 286-185 286-157 289-185 0.95

Codeine 300-165 300-215 303-165 1.28

Oxycodone 316-241 316-298 319-244 1.35

Hydrocodone 300-199 300-128 303-199 1.4

Noroxycodone 302-284 302-227 1.42

Amphetamine 136-91 136-119 144-97 1.43

Norhydrocodone 286-199 286-128 1.43

MDA 180-163 180-105 185-168 1.49

Methamphetamine 151-92 151-120 159-93 1.49

6 MAM 328-165 328-211 334-165 1.5

MDMA 194-163 194-105 199-165 1.53

O-Desmethyl

Tramadol

250-58 232.2 256-64 1.61

MDEA 208-163 208-105 213-163 1.68

7-Amino

Clonazepam

286-121 286-222 290-121 1.74

BZE 290-168 290-105 293-171 1.77

Norfentanyl 233-150 233-177 238-155 1.77

Tramadol 264-58 264-246 268-58 1.81

Cocaine 304-182 304-105 307-185 1.82

Tapentadol 222-107 222-121 225-107 1.85

7-Amino

Flunitrazepam

284-135 284-240 291-138 1.87

Meperidine 248-220 248-174 252-224 1.88

Normeperidine 234-160 234-91 238-164 1.92

PCP 245-91 245-160 250-96 1.95

Drug Quant Confirm IS

RT

(min)

Fentanyl 337-188 337-105 342-105 1.97

Norbuprenorphine 414-187 414-101 417-55 1.97

EDDP 278-234 278-249 281-234 2.08

Flurazepam 388-315 388-317 2.1

Midazolam 326-291 326-249 330-295 2.1

Meprobamate 219-158 219-97 226-165 2.13

Buprenorphine 468-396 468-414 472-59 2.2

Chlordiazepoxide 300-227 300-282 305-232 2.3

Alpha-Hydroxy

Midazalam 342-324 342-203 346-328 2.38

Propoxyphene 340-58 340-266 351-64 2.45

Methadone 310-265 310-105 319-105 2.59

Nitrazepam 282-236 282-180 287-241 2.62

Norpropoxyphene 326-252 326-91 331-313 2.5

Clonazepam 316-270 316-214 320-274 2.7

Alpha-Hydroxy

Triazolam 359-331 359-176 363-335 2.76

Flunitrazepam 314-268 314-239 321-275 2.77

Alpha-Hydroxy

Alprazolam 325-297 325-216 330-302 2.87

2-Hydroxyethyl

Flurazepam 333-109 333-211 337-113 3

Carisoprodol 261-176 261-97 268-183 3

Lorazepam 321-275 321-303 325-279 3.01

Oxazepam 287-241 287-269 292-246 3.02

Alprazolam 309-281 309-205 314-286 3.06

Temazepam 301-255 301-283 306-260 3.2

Nordiazepam 271-140 271-208 276-140 3.33

Diazepam 285-193 285-154 290-198 3.54

Prazepam 325-271 325-140 330-276 3.85

Results

Standards of the drugs of interest were prepared in

urine over the range of 10-5000 ng/mL, (except for

normeperidine, buprenorphine, and norbuprenorphine

which were 1-500 ng/mL) internal standard was added,

samples were hydrolyzed and then processed with the

ITSP cleanup procedure prior to analysis by LC-MS/MS.

The calibration curves that were obtained are shown

below, plotting the analyte to internal standard area

ratio versus the concentration (ng/mL).







Representative chromatograms of each class are

shown below. The remainder are available upon

request.





Determination of Barbiturates and THCA in Human Urine

Using Automated ITSP Solid Phase Extraction and

Liquid Chromatography Mass Spectrometric Detection

Thurman L. Allsup1, Kenneth C. Lewis

1, Rick Youngblood

2 and Kim Gamble

2

1OpAns, LLC, Durham NC, 27713,

2MicroLiter Analytical Supplies, Inc., Suwannee GA, 30024

Abstract

A quantitative analytical method has been developed

for the determination of phenobarbital, butabarbital,

butalbital, secobarbital, amobarbital, pentobarbital and

11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic acid

(THCA) in human urine. The method that has been

developed here is fully automated to quantitatively

extract barbiturates and THCA from urine using QAX

(strong anion exchange) ITSP SPE cartridges 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 System (CTC Analytics)

sample handling unit. Spiked urine samples with stable

label internal standards were adjusted to basic pH with

pH 10.5 buffer and then applied to QAX ITSP cartridges

for extraction. The extracts were injected in-line onto

an LC/MS/MS using a XTerra MS C18, 3.5 µm, 2.1x50 mm

column for separation. The extracted standard curve

had a range of 0.1 - 20 µg/mL for the barbiturates and

0.01 – 2 µg/mL for THCA. The correlation coefficients

were greater than 0.99 for all compounds. Selected

reaction monitoring (SRM) was used for the

quantitation.

Experimental

Sample Preparation

Phenobarbital, butabarbital, butalbital, secobarbital,

pentobarbital, amobarbital, and THCA standards and the

deuterated analogs of butalbital, secobarbital, phenobarbital,

pentobarbital and THCA were obtained from Cerillant. A set

of standards was prepared in human urine. Calibration

standards were prepared at nominal concentrations of 0.1,

0.5, 1, 10, and 20 µg/mL with a constant concentration of the

deuterated analog at 1 µg/mL for the barbiturates.

Calibration standards were prepared at nominal

concentrations of 0.01, 0.05, 0.1, 1.0, and 2.0 µg/mL with a

constant concentration of the deuterated analog at 0.1 µg/mL

buffer.

BUTALBITAL

EXACT MASS 224.2

BUTABARBITAL

EXACT MASS 212.2

PHENOBARBITAL

EXACT MASS 232.2

SECOBARBITAL

EXACT MASS 238.3

PENTOBARBITAL

EXACT MASS 226.2

AMOBARBITAL

EXACT MASS 226.2

THCA

EXACT MASS 344.2



ITSP SPE Method

ITSP Cartridges: UCT SelectrasorbTM

QAX 10mg

(Product No.: 07-UQAX10-20A)

A CTC Analytics HTS PAL sample handler was used to prepare

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

µL)

Flowrate

µL/sec)

Load Sample 500 10

Wash A 100 10

Wash B 100 10

Flush Air 100 100

Elute C 100 5

Flush Air 100 100

Solvent A: 100mM Carbonate pH 10.5 buffer

Solvent B: 1% Ammonium hydroxide in methanol

Solvent C: 5% Acetic acid in methanol

Samples were prepared and analyzed in-line with the

following method:

Analysis Method

Instrument: Finnigan TSQ Quantum Ultra AM triple

quadrupole with Agilent 1200 Rapid Resolution

HPLC

Column: XTerra MS C18, 2.1x50 mm, 3.5 µm particles

Solvent A: Water with 5 mM ammonium acetate and

0.05% ammonium hydroxide

Solvent B: Methanol with 0.05% ammonium hydroxide

Column Temp.: Ambient

Flowrate: 0.5 mL/min

Gradient: 0.00 min (10% B), 0.5 min (10% B), 4.0 min

(40% A), 5.6 min (100% B), 5.8 min (100% B),

6.0 min (10% B)

Ionization Mode: Negative Ion (HESI) Heated Electrospray

Ionization Probe

SRM Channels:

Quant. Confirm IS

Butabarbital 211-211 211-42 *

Butalbital 223-223 223-42 228-228

Secobarbital 237-237 237-42 242-242

Phenobarbital 231-231 231-42 236-236

Amobarbital 225-225 225-42 **

Pentobarbital 225-225 225-42 230-230

THCA 343-299 343-245 346-302

* Use Phenobarbital IS ** Use Pentobarbital IS

Results

Standards of the six barbiturates and THCA were prepared in

urine over the range of 0.1 - 20 µg/mL and 0.01 – 2.0 µg/mL,

respectively, and processed in-line with an ITSP cleanup

procedure followed by LC-MS/MS. The calibration curves

that were obtained are shown below, plotting the analyte to

internal standard area ratio versus the concentration of the

analyte. Because of the barbiturates extremely poor

fragmentation, the precursor to precursor SRM transition

was used for all barbiturates and the confirmatory SRM

transition was only useful for the higher concentrations.

Butalbital

Y = 0.00935706+0.84444*X R^2 = 0.9994 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Are

a R

atio

Ratio

Phenobarbital

Y = -0.0464945+1.08484*X R^2 = 0.9989 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

Are

a R

atio

Ratio



Conclusions

A simplified, automated procedure for the analysis of

butabarbital, butalbital, secobarbital, phenobarbital,

pentobarbital, amobarbital and THCA in human urine

has been developed using ion exchange ITSP SPE for

in-line sample cleanup and LC-MS/MS for detection. A

linear response was observed over the concentration

range of 0.1 - 20 µg/mL for all of the barbiturates and

over the range of 0.01 - 2 µg/mL for THCA.

THCA

Y = -0.0222865+7.79162*X R^2 = 0.9945 W: 1/X

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Are

a R

atio

Ratio

Secobarbital

Y = -0.0401594+0.815851*X R^2 = 0.9906 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Are

a R

atio

Ratio

Pentobarbital

Y = -0.0418638+0.883712*X R^2 = 0.9919 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Are

a R

atio

Ratio

Amobarbital

Y = 0.0936486+0.810262*X R^2 = 0.9913 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Are

a R

atio

Ratio

Butabarbital

Y = -0.00965978+0.953161*X R^2 = 0.9997 W: 1/X

0 2 4 6 8 10 12 14 16 18 20 22 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Are

a R

atio

Ratio



ITSP – Pain Management Drugs and Drugs of Abuse


www.microliter.com Rev Date: 101220

888-232-7840 • 770-932-6565

www.microliter.com

PO Box 808, Suwanee, GA 30024

ITSP Components by MicroLiter:

Part Number Description

07-ITSP-HW

07-UDBX10-20A

07-UQAX10-20A

Hardware kit to modify CTC PAL for use with ITSP

ITSP SPE cartridge – packed with 10mg of UCT Styre Screen DBX

ITSP SPE cartridge – packed with 10mg of UCT SelectrasorbTM

QAX

The information in this document is subject to change without notice.

MicroLiter Analytical Supplies, Inc. makes no warranty of any kind with regard to this material, including, but not limited to, the

implied warranties or merchantability and fitness for a particular purpose.

MicroLiter Analytical Supplies, Inc. shall not be liable for errors contained herein or for incidental or consequential damages in

connection with the furnishing, performance, or use of this material.

Contents of this publication may not be reproduced in any form or by any means (including electronic storage and retrieval or

translation into a foreign language) without the prior agreement and written consent from the copyright owner.

© 2009 MicroLiter Analytical Supplies, Inc. All rights reserved.

ITSP™

is protected under the following US Patents 6,859,615 & 7,001,774, Canadian Patent 2,316,648, and European Block Patent EP 1 174 701.

Other US and Foreign Patents Pending.

ITSP™ and The MicroLiter Plate Sampling System are trademarks of MicroLiter Analytical Supplies, Inc.

The logo, MicroLiter and MicroLiter Analytical Supplies, Inc. are registered trademarks of MicroLiter Analytical Supplies, Inc.

All rights reserved.

http://www.microliter.com/

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