APPLICATIONSTN-1141

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iMethod Test for LC/MS/MS Analysis of Pain Management Drugs (Pain Panel) from Human Urine using a Kinetex® 2.6 µm Phenyl-Hexyl HPLC/UHPLC Column

Using a Kinetex 2.6 µm Core-Shell Technology HPLC/UHPLC column, a large panel of 41 pain management drugs was analyzed from human urine in under 5.5 minutes.

Materials and Methods Prior to sample preparation, internal standard spiking solution, calibration curve diluent, and β-Glucuronidase solution must be prepared as follows:

Internal Standard Preparation: Prepare the combined internal standard spiking solution as follows:

1. Add 4.084 mL of water to make a final volume of 5 mL with concentrations as outlined in Table 1.

2. Prepare in a glass vial and store refrigerated.

Calibration Curve Stock Preparation (CCSS): Prepare the combined CCSS as follows:

1. Mix 100 µL of the 1000 µg/mL stock with 900 µL of methanol to prepare a 100 µg/mL substock solution of 6-MAM.

2. Mix 10 µL of the 1000 µg/mL stock with 990 µL of methanol to prepare a 10 µg/mL substock solution of fentanyl.

3. Mix 100 µL of the 1000 µg/mL stock with 900 µL of methanol to prepare a 100 µg/mL substock solution of norfentanyl.

4. Mix 100 µL of the 1000 µg/mL stock with 900 µL of methanol to prepare a 100 µg/mL substock solution of PCP.

5. Mix 100 µL of the 100 µg/mL stock with 900 µL of methanol to prepare a 10 µg/mL substock solution of sufentanil.

6. Store at -20 °C.

7. Add 15 µL of methanol to make a final volume of 1 mL with concentrations as outlined in Table 2.

8. Store refrigerated

Internal Standards

6-Acetylmorphine-d3 (6-MAM-d3) Methadone-d3

Amphetamine-d5 Methamphetamine-d5

Benzoylecgonine-d3 Morphine-d6

Codeine-d6 Nordiazepam-d5

Fentanyl-d5 Normeperidine-d4

Hydrocodone-d6 Norpropoxyphene-d5

Hydromorphone-d6 Phencyclidine-d5

Meperidine-d4

Analytes

6-Acetylmorphine (6-MAM) Methadone

α-Hydroxyalprazolam Methamphetamine

Alprazolam Midazolam

Amphetamine Morphine

Benzoylecgonine Naloxone

Buprenorphine Naltrexone

Carisoprodol Norbuprenorphine

Clonazepam Nordiazepam

Codeine Norfentanyl

Diazepam Normeperidine

EDDP Norpropoxyphene

Fentanyl Oxazepam

Flunitrazepam Oxycodone

Flurazepam Oxymorphone

Hydrocodone Phencyclidine (PCP)

Hydromorphone Propoxyphene

Lorazepam Sufentanil

MDA Temazepam

MDEA Tramadol

MDMA Temazepam

Meperidine Tramadol

Meprobamate

Table 1.

Internal StandardStock

Concentration (µg/mL)

Spike Volume

(µL)

Resulting Concentration

(ng/mL)

6-MAM-d3 100 30 600

Amphetamine-d5 100 125 2500

Benzoylecgonine-d3 100 37.5 750

Codeine-d6 100 25 500

Fentanyl-d5 100 5 100

Hydrocodone-d6 100 25 500

Hydromorphone-d6 100 25 500

Meperidine-d4 100 150 3000

Methadone-d3 100 25 500

Methamphetamine-d5 100 125 2500

Morphine-d6 100 25 500

Nordiazepam-d5 100 25 500

Normeperidine-d4 100 25 500

Norpropoxyphene maleate-d5 100 37.5 750

Phencyclidine-d5 100 6.25 125

For AB SCIEX mass spectrometer users, this method can be instantly implemented by installing the iMethod™ Test. iMethod Tests are verified across several laboratories and

contain everything you will need to start running samples including sample preparation recommendations, consumables, LC running conditions, optimized MRM parameters, reporting templates, and complete method documentation.

Visit www.phenomenex.com/iMethod for more information about available iMethod Tests.

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Table 2.

AnalyteStock

Concentration (µg/mL)

Spike Volume

(µL)

Resulting Concentration

(ng/mL)

6-MAM 100 10 1000

α-Hydroxyalprazolam 1000 10 10000

Alprazolam 1000 10 10000

Amphetamine 1000 50 50000

Benzoylecgonine 1000 15 15000

Buprenorphine 100 100 10000

Carisoprodol 1000 20 20000

Clonazepam 1000 10 10000

Codeine 1000 10 10000

Diazepam 1000 10 10000

EDDP 1000 10 10000

Fentanyl 10 30 300

Flunitrazepam 1000 10 10000

Flurazepam 1000 10 10000

Hydrocodone 1000 10 10000

Hydromorphone 1000 10 10000

Lorazepam 1000 10 10000

MDA 1000 25 25000

MDEA 1000 25 25000

MDMA 1000 25 25000

Meperidine 1000 100 100000

Meprobamate 1000 20 20000

Methadone 1000 10 10000

Methamphetamine 1000 50 50000

Midazolam 1000 10 10000

Morphine 1000 10 10000

Naloxone 1000 10 10000

Naltrexone 1000 10 10000

Norbuprenorphine 100 100 10000

Nordiazepam 1000 10 10000

Norfentanyl 100 10 1000

Normeperidine 100 100 10000

Norpropoxyphene 1000 15 15000

Oxazepam 1000 10 10000

Oxycodone 1000 10 10000

Oxymorphone 1000 10 10000

PCP 100 25 2500

Propoxyphene 1000 15 15000

Sufentanil 10 30 300

Temazepam 1000 10 10000

Tramadol 1000 10 10000

Table 3.

Calibration Standard

Solutions Used

Spike Volume (µL)

Urine Volume (µL)

Final Volume (mL)

10x Cutoff CCSS 50 450 0.500

5x Cutoff CCSS 25 475 0.500

3x Cutoff CCSS 15 485 0.500

Cutoff 10x Cutoff 50 450 0.500

75 % Cutoff 10x Cutoff 37.5 462.5 0.500

60 % Cutoff 10x Cutoff 30 470 0.500

40 % Cutoff 10x Cutoff 20 480 0.500

Calibration Curve Preparation: The calibration curve is prepared in a urine:10 % methanol solution as follows:

1. Mix 18 mL of synthetic urine (surine) with 2 mL of methanol.

2. Store refrigerated.

3. Prepare calibration standards in glass vials as outlined in Table 3.

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β-Glucuronidase Solution Preparation:

1. Dissolve the β-Glucuronidase powder (1,000,000 units) in 10 mL of water.

2. Store refrigerated.

Note: The resulting solution has a concentration of 100,000 units/mL.

General method for urine, calibrator, and controls- no hydrolysis of samples

1. Measure 50 µL of the urine sample into a 2 mL glass amber autosampler vial

2. Add 20 µL of the combined internal standard spiking solution to each vial and then mix using a vortex mixer for approxmately 5 to 10 seconds

3. Add 150 µL of the curve diluent to each calibration curve sample and mix using a vortex mixer for approximately one minute

4. Add 150 µL of the sample diluent to each study sample and then mix using a vortex mixer for approximately one minute

5. Transfer the sample to a small volume HPLC vial

6. Analyze samples by LC/MS/MS

General method for urine study sample, calibrator and controls- with hydrolysis

1. Measure 50 µL of the urine sample into a 2 mL glass amber autosampler vial

2. Add 20 µL of the combined internal standard spiking solution to each vial and then mix using a vortex mixer for approximately 5 to 10 seconds

3. Add 25 µL of 0.1 M Acetate buffer (pH 4.0) to each sample and then mix using a vortex mixer for 15 seconds

4. Add 10 µL of β-Glucuronidase solution (100,000 units/mL) to each sample and then mix using a vortex mixer for 15 seconds

5. Incubate for 2 hours in a shaking water bath at 55 °C to complete hydrolysis of the glucuronides

6. Add 110 µL of the curve diluent to each study sample and then mix using a vortex mixer for approximately one minute

7. Centrifuge the hydrolyzed samples at approximately 21,000 g for 10 minutes to separate any proteins

8. Transfer the supernatant to a small volume HPLC vial

9. Analyze samples by LC/MS/MS

LC/MS/MS ConditionsColumn: Kinetex® 2.6 µm Phenyl-Hexyl

Dimensions: 50 x 4.6 mmPart No.: 00B-4495-E0

Guard: SecurityGuard™ ULTRA UHPLC PhenylGuard Part No.: AJ0-8774 Mobile Phase: A: 10 mM Ammonium formate

B: 0.1 % Formic acid in MethanolFlow Rate: 0.7 mL/min

Gradient:Time (min) % B

0.01 52.20 404.50 955.50 955.60 5

Injection Volume: 25 µLTemperature: 30 °C

Detection: API 3200™ or

API 4000™ MS/MS, positive polarity

Resulting Concentrations:

Analyte 40 % Cutoff

60 % Cutoff

75 % Cutoff

Cutoff 3x Cutoff

5x Cutoff

10x Cutoff

6-MAM 4 6 7.5 10 30 50 100

α-Hydroxyalprazolam 40 60 75 100 300 500 1000

Alprazolam 40 60 75 100 300 500 1000

Amphetamine 200 300 375 500 1500 2500 5000

Benzoylecgonine 60 90 112.5 150 450 750 1500

Buprenorphine 40 60 75 100 300 500 1000

Carisoprodol 80 120 150 200 600 1000 2000

Clonazepam 40 60 75 100 300 500 1000

Codeine 40 60 75 100 300 500 1000

Diazepam 40 60 75 100 300 500 1000

EDDP 40 60 75 100 300 500 1000

Fentanyl 1.2 1.8 2.25 3 9 15 30

Flunitrazepam 40 60 75 100 300 500 1000

Flurazepam 40 60 75 100 300 500 1000

Hydrocodone 40 60 75 100 300 500 1000

Hydromorphone 40 60 75 100 300 500 1000

Lorazepam 40 60 75 100 300 500 1000

MDA 100 150 187.5 250 750 1250 2500

MDEA 100 150 187.5 250 750 1250 2500

MDMA 100 150 187.5 250 750 1250 2500

Meperidine 400 600 750 1000 3000 5000 10000

Meprobamate 80 120 150 200 600 1000 2000

Methadone 40 60 75 100 300 500 1000

Methamphetamine 200 300 375 500 1500 2500 5000

Midazolam 40 60 75 100 300 500 1000

Morphine 40 60 75 100 300 500 1000

Naloxone 40 60 75 100 300 500 1000

Naltrexone 40 60 75 100 300 500 1000

Norbuprenorphine 40 60 75 100 300 500 1000

Nordiazepam 40 60 75 100 300 500 1000

Norfentanyl 4 6 7.5 10 30 50 100

Normeperidine 40 60 75 100 300 500 1000

Norpropoxyphene 60 90 112.5 150 450 750 1500

Oxazepam 40 60 75 100 300 500 1000

Oxycodone 40 60 75 100 300 500 1000

Oxymorphone 40 60 75 100 300 500 1000

PCP 10 15 18.75 25 75 125 250

Propoxyphene 60 90 112.5 150 450 750 1500

Sufentanil 1.2 1.8 2.25 3 9 15 30

Temazepam 40 60 75 100 300 500 1000

Tramadol 40 60 75 100 300 500 1000

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MS/MS Detection

Optimized for API 3200™, 3200 QTRAP®, API 4000™, and 4000 QTRAP LC/MS/MS

TurboIonSpray® ion source

Positive polarity

Source/Gas Parameters

CUR: 20 psi

IS: 2500

TEM: 650 °C

GS1: 60 psi

GS2: 50 psi

Ihe: On

CAD: 6 (medium on 3200 QTRAP and 4000

QTRAP LC/MS/MS system)

Compound MRM Parameters and Retention Times

DP: variable

EP: variable

CXP: variable

Dwell Time: Scheduled MRM™ algorithm with set

retention times

Target scan time: 0.4 s

Test injections using unscheduled MRM should be evaluated to determine the correct retention times for the system used.

Q1 Resolution: UNIT

Q3 Resolution: UNIT

Compound MRM Parameters for API 3200 MS/MS System

Analyte Q1(m/z)

Q3(m/z)

DP(V)

EP(V)

CE(V)

CXP(V)

6-MAM 1 328.1 165.2 61 7 47 4

6-MAM 2 328.1 211.2 61 7 33 4

Alprazolam 1 309.1 281 56 7 37 4

Alprazolam 2 309.1 205.1 56 7 55 4

Amphetamine 1 136.1 91.0 51 7 23 4

Amphetamine 2 136.1 119.0 51 7 11 4

Benzoylecgonine 1 290.2 168.1 41 7 25 4

Compound MRM Parameters for API 3200 MS/MS System con’t

Analyte Q1(m/z)

Q3(m/z)

DP(V)

EP(V)

CE(V)

CXP(V)

Buprenorphine 2 468.3 414.2 81 11 43 6

Carisoprodol 1 261.2 176.2 26 8 17 4

Carisoprodol 2 261.2 97.2 26 8 23 4

Clonazepam 1 316.0 270.1 56 11 31 2

Clonazepam 2 316.0 214.2 56 11 49 2

Codeine 1 300.1 152.1 56 9 83 4

Codeine 2 300.1 115.1 56 9 97 4

Diazepam 1 285.1 193.1 46 10 41 4

Diazepam 2 285.1 154.0 46 10 35 4

EDDP 1 278.3 234.2 100 9 57 4

EDDP 2 278.3 186.2 100 9 43 4

Fentanyl 1 337.2 105.1 51 9 53 4

Fentanyl 2 337.2 188.1 51 9 29 4

Flunitrazepam 1 314.1 268.2 56 9 33 4

Flunitrazepam 2 314.1 239.2 56 9 45 4

Flurazepam 1 388.2 315.1 46 8.5 29 6

Flurazepam 2 388.2 134.1 46 8.5 71 4

Hydrocodone 1 300.1 199.1 56 8 39 4

Hydrocodone 2 300.1 128.1 56 8 69 4

Hydromorphone 1 286.2 185.0 56 10 39 4

Hydromorphone 2 286.2 128.0 56 10 73 4

Hydroxyalprazolam 1 325.1 297.0 51 11 33 6

Hydroxyalprazolam 2 325.1 216.1 51 11 53 4

Lorazepam 1 321.0 275.1 81 9.5 23 4

Lorazepam 2 321.0 229.1 81 9.5 41 4

MDA 1 180.1 105.0 16 5.5 29 4

MDA 2 180.1 133.0 16 5.5 23 4

MDEA 1 208.2 163.2 55 6 17 4

MDEA 2 208.2 105.2 55 6 31 4

MDMA 1 194.1 163.2 50 7.5 17 4

MDMA 2 194.1 105.2 50 7.5 31 4

Meperidine 1 248.2 220.0 95 5.5 43 4

Meperidine 2 248.2 174.1 95 5.5 29 4

Meprobamate 1 219.2 158.2 41 7 13 4

Meprobamate 2 219.2 97.1 41 7 17 4

Methadone 1 310.2 265.2 31 7.5 19 4

Methadone 2 310.2 105.0 31 7.5 43 4

Methamphetamine 1 150.1 91.2 50 7.5 27 4

Methamphetamine 2 150.1 119.2 50 7.5 15 4

Midazolam 1 326.1 291.1 56 9.5 33 6

Midazolam 2 326.1 249.1 56 9.5 47 4

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Compound MRM Parameters for API 3200 MS/MS System con’t

Analyte Q1(m/z)

Q3(m/z)

DP(V)

EP(V)

CE(V)

CXP(V)

Methamphetamine-d5 155.2 92.0 26 5.5 27 4

Morphine-d6 292.2 152.1 66 10.5 77 4

Nordiazepam-d5 276.1 140.0 61 8 39 4

Normeperidine-d4 238.2 164.2 36 8.5 23 4

Norpropoxyphene-d5 313.2 100.1 26 6.5 17 4

PCP-d5 249.3 96.0 21 5.5 41 4

Transition 1 for each analyte is used as the quantifier transition

Transition 2 for each analyte is used as the qualifier transition

Compound MRM Parameters for API 3200™ MS/MS System con’t

Analyte Q1(m/z)

Q3(m/z)

DP(V)

EP(V)

CE(V)

CXP(V)

Morphine 1 286.2 152.0 56 9.5 73 4

Morphine 2 286.2 165.0 56 9.5 47 4

Naloxone 1 328.1 212.0 46 7.5 47 4

Naloxone 2 328.1 253.1 46 7.5 35 4

Naltrexone 1 342.2 267.1 41 8 37 4

Naltrexone 2 342.2 212.1 41 8 57 4

Norbuprenorphine 1 414.3 101.1 76 6.5 53 4

Norbuprenorphine 2 414.3 165.1 76 6.5 103 4

Nordiazepam 1 271.1 140.0 56 8 37 4

Nordiazepam 2 271.1 165.1 56 8 37 4

Norfentanyl 1 233.2 84.1 41 5.5 23 4

Norfentanyl 2 233.2 150.0 41 5.5 23 4

Normeperidine 1 231.1 160.2 36 7 21 4

Normeperidine 2 234.1 188.1 36 7 17 4

Norpropoxyphene 1 308.2 100.1 31 7.5 17 4

Norpropoxyphene 2 308.2 143.2 31 7.5 29 4

Oxazepam 1 287.1 241.0 41 6.5 27 6

Oxazepam 2 287.1 269.1 41 6.5 19 4

Oxycodone 1 316.2 241.0 46 7 37 4

Oxycodone 2 316.2 256.0 46 7 33 4

Oxymorphone 1 302.1 227.0 41 12 37 4

Oxymorphone 2 302.1 198.1 41 12 55 4

PCP 1 244.3 91.0 21 7 41 4

PCP 2 244.3 159.3 21 7 19 4

Propoxyphene 1 340.2 266.3 16 6 15 6

Propoxyphene 2 340.2 91.1 16 6 67 4

Sufentanil 1 387.2 238.2 36 5.5 25 4

Sufentanil 2 387.2 111.0 36 5.5 49 4

Temazepam 1 301.1 255.1 31 8 27 4

Temazepam 2 301.1 177.1 31 8 55 4

Tramadol 1 264.2 58.1 60 7.5 75 8

Tramadol 2 264.2 42.1 60 7.5 107 8

6-MAM-d3 331.1 165.0 50 7 30 12

Amphetamine-d5 141.1 93.0 21 5.5 23 4

Benzoylecgonine-d3 293.2 171.2 41 8.5 25 4

Codeine-d6 306.2 152.1 61 9 81 4

Fentanyl-d5 342.2 105.1 46 11.5 53 4

Hydrocodone-d6 306.2 202.1 96 8 53 12

Hydromorphone-d6 292.1 185.1 126 10 41 16

Meperidine-d4 252.2 224.2 46 8 29 4

Methadone-d3 313.2 105.0 31 6.5 35 4

Compound MRM Parameters for API 4000™ MS/MS System

Analyte Q1(m/z)

Q3(m/z)

DP(V)

CE(V)

CXP(V)

6-MAM 1 328.1 165.1 81 59 10

6-MAM 2 328.1 211.1 81 37 12

Alprazolam 1 309.1 281.1 116 47 22

Alprazolam 2 309.1 205.2 116 71 12

Amphetamine 1 136.1 91.0 41 60 16

Amphetamine 2 136.1 119.0 41 25 6

Benzoylecgonine 1 290.1 168.1 86 43 10

Benzoylecgonine 2 290.1 105.0 86 71 6

Buprenorphine 1 468.3 396.2 116 55 12

Buprenorphine 2 468.3 414.3 116 45 10

Carisoprodol 1 261.1 176.2 46 21 10

Carisoprodol 2 261.1 97.2 46 37 4

Clonazepam 1 316.1 270.1 131 48 18

Clonazepam 2 316.1 214.0 131 53 12

Codeine 1 300.2 152.1 106 63 10

Codeine 2 300.2 115.1 106 75 20

Diazepam 1 285.0 193.2 101 66 10

Diazepam 2 285.0 154.1 101 57 8

EDDP 1 278.2 234.2 86 65 14

EDDP 2 278.2 186.2 86 72 10

Fentanyl 1 337.3 105.1 91 80 18

Fentanyl 2 337.3 188.2 91 42 12

Flunitrazepam 1 314.1 268.2 91 50 22

Flunitrazepam 2 314.1 239.2 91 62 14

Flurazepam 1 388.2 315.2 71 54 20

Flurazepam 2 388.2 134.1 71 101 6

Hydrocodone 1 300.2 199.0 96 53 12

Hydrocodone 2 300.2 128.0 96 99 6

Hydromorphone 1 286.1 185.1 126 41 16

Hydromorphone 2 286.1 128.0 126 77 6

Hydroxyalprazolam 1 325.1 297.0 111 35 20

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Compound MRM Parameters for API 4000™ MS/MS System con’t

Analyte Q1(m/z)

Q3(m/z)

DP(V)

CE(V)

CXP(V)

Lorazepam 2 321.0 229.0 91 43 14

MDA 1 180.1 133.0 46 35 8

MDA 2 180.1 105.0 46 47 8

MDEA 1 208.2 163.0 66 35 10

MDEA 2 208.2 105.0 66 63 18

MDMA 1 194.1 105.1 61 59 6

MDMA 2 194.1 163.0 61 35 10

Meperidine 1 248.2 220.2 91 48 36

Meperidine 2 248.2 174.2 91 61 12

Meprobamate 1 219.2 158.1 51 13 8

Meprobamate 2 219.2 97.0 51 21 16

Methadone 1 310.0 265.0 106 34 26

Methadone 2 310.0 105.0 106 74 6

Methamphetamine 1 150.1 91.0 70 69 16

Methamphetamine 2 150.1 119.0 70 29 6

Midazolam 1 326.1 291.1 111 53 18

Midazolam 2 326.1 249.1 111 64 16

Morphine 1 286.1 152.1 91 89 8

Morphine 2 286.1 165.1 91 51 10

Naloxone 1 328.2 212.0 86 53 12

Naloxone 2 328.2 253.0 86 37 16

Naltrexone 1 342.2 267.1 96 57 18

Naltrexone 2 342.2 212.1 96 76 12

Norbuprenorphine 1 414.3 101.1 146 53 18

Norbuprenorphine 2 414.3 165.2 146 103 8

Nordiazepam 1 271.0 140.0 116 39 8

Nordiazepam 2 271.0 165.1 116 37 10

Norfentanyl 1 233.2 84.1 46 25 16

Norfentanyl 2 233.2 150.1 46 25 12

Normeperidine 1 234.1 160.1 71 35 10

Normeperidine 2 234.1 188.1 71 25 16

Norpropoxyphene 1 308.2 100.1 41 36 4

Norpropoxyphene 2 308.2 143.2 41 48 8

Oxazepam 1 287.0 241.0 106 33 14

Oxazepam 2 287.0 269.1 106 21 18

Oxycodone 1 316.1 241.2 91 37 22

Oxycodone 2 316.1 256.2 91 33 16

Oxymorphone 1 302.1 227.1 101 41 14

Oxymorphone 2 302.1 198.2 101 61 10

PCP 1 244.3 91.1 55 72 16

PCP 2 244.3 159.2 55 28 8

Compound MRM Parameters for API 4000 MS/MS System con’t

Analyte Q1(m/z)

Q3(m/z)

DP(V)

CE(V)

CXP(V)

Propoxyphene 1 340.3 266.3 41 18 20

Propoxyphene 2 340.3 91.1 41 53 16

Sufentanil 1 387.2 238.1 75 25 15

Sufentanil 2 387.2 111.1 75 54 20

Temazepam 1 301.1 255.1 81 51 6

Temazepam 2 301.1 177.1 81 65 10

Tramadol 1 264.1 58.1 56 108 10

Tramadol 2 264.1 42.1 56 109 6

6-MAM-d3 331.1 165.0 50 30 12

Amphetamine-d5 141.1 93.0 46 59 16

Benzoylecgonine-d3 293.1 171.2 81 43 10

Codeine-d6 306.2 152.2 106 117 8

Fentanyl-d5 342.3 105.1 91 83 6

Hydrocodone-d6 306.2 202.1 96 53 12

Hydromorphone-d6 292.1 185.1 126 41 16

Meperidine-d4 252.2 224.1 96 48 14

Methadone-d3 313.2 105.1 56 72 6

Methamphetamine-d5 155.2 92.0 51 74 16

Morphine-d6 292.1 152.0 96 83 8

Nordiazepam-d5 276.1 140.0 86 55 8

Normeperidine-d4 238.2 164.2 76 33 10

Norpropoxyphene-d5 313.2 100.1 56 37 6

PCP-d5 249.3 96.1 41 76 4

Transition 1 for each analyte is used as the quantifier transition

Transition 2 for each analyte is used as the qualifier transition

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Analyte Internal Standard

6-MAM 6-MAM-d3

α-Hydroxyalprazolam Nordiazepam-d5

Alprazolam Nordiazepam-d5

Amphetamine Amphetamine-d5

Benzoylecgonine Benzoylecgonine-d3

Buprenorphine Nordiazepam-d5

Carisoprodol Fentanyl-d5

Clonazepam Nordiazepam-d5

Codeine Codeine-d6

Diazepam Nordiazepam-d5

EDDP Norpropoxyphene-d5

Fentanyl Fentanyl-d5

Flunitrazepam Nordiazepam-d5

Flurazepam Nordiazepam-d5

Hydrocodone Hydrocodone-d6

Hydromorphone Hydromorphone-d6

Lorazepam Nordiazepam-d5

MDA Methamphetamine-d5

MDEA Benzoylecgonine-d3

MDMA Benzoylecgonine-d3

Meperidine Meperidine-d4

Meprobamate Benzoylecgonine-d3

Methadone Methadone-d3

Methamphetamine Methamphetamine-d5

Midazolam Nordiazepam-d5

Morphine Morphine-d6

Naloxone Codeine-d6

Naltrexone 6-MAM-d3

Norbuprenorphine Meperidine-d4

Nordiazepam Nordiazepam-d5

Norfentanyl Benzoylecgonine-d3

Normeperidine Normeperidine-d4

Norpropoxyphene Norpropoxyphene-d5

Oxazepam Nordiazepam-d5

Oxycodone Codeine-d6

Oxymorphone Morphine-d6

PCP PCP-d5

Propoxyphene Norpropoxyphene-d5

Sufentanil Nordiazepam-d5

Temazepam Nordiazepam-d5

Tramadol Normeperidine-d4

Chromatogram of Calibration Standard at 3x Cutoff Overlaid MRM ion traces for the quantifier MRM transitions, performed on a 3200 QTRAP® system with a Shimadzu® Prominence® LC system in a 6.5 minute run

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 min0.0

1.0e52.0e53.0e54.0e55.0e56.0e57.0e58.0e59.0e51.0e61.1e61.2e61.3e61.4e61.5e61.6e61.7e61.8e61.9e62.0e62.1e62.2e62.3e62.4e6

Inte

nsity

, cp

s

Ap

p ID

208

52

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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0

4.0e4

5.0e4

6.0e4

7.0e4

1.0e4

2.0e4

3.0e4

8.0e4

9.0e4

1.0e5

1.1e5

Inte

nsity

, cp

s

0.50

5.0e41.0e51.5e52.0e52.5e53.0e53.5e54.0e54.5e55.0e55.5e56.0e56.5e5

Inte

nsity

, cp

s

6.8e5

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min

Individual Representative Chromatograms of Spike Urine Samples at 3x Cutoff Level

0.50

2.0e44.0e46.0e48.0e41.0e51.2e51.4e51.6e51.8e52.0e52.2e52.4e52.6e52.8e53.0e53.2e5

Inte

nsity

, cp

s

3.4e5

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min

Benzoylecgonine

Diazepam

a-Hydroxyalprazolam

APPLICATIONSTN-1141

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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0

1.0e42.0e43.0e44.0e4

5.0e46.0e47.0e48.0e4

9.0e4

1.1e5

1.0e5

1.2e51.3e5

Inte

nsity

, cp

s

1.4e5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min

1.0e40

2.0e43.0e44.0e4

5.0e46.0e47.0e48.0e49.0e4

1.1e51.0e5

1.2e51.3e5

Inte

nsity

, cp

s

1.4e5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0

4.0e43.0e42.0e41.0e4

5.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e5

Inte

nsity

, cp

s

1.6e5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 min0

2.0e4

4.0e56.0e5

8.0e51.0e5

1.2e51.4e51.6e51.8e5

2.0e5

2.2e5

Inte

nsity

, cp

s

2.4e5MDMA Propoxyphene

Lorazepam Methamphetamine

APPLICATIONSTN-1141

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Representative Accuracy and Precision in Spiked Urine

Analyte 40 % Cutoff

60 % Cutoff

75 % Cutoff

Cutoff 3x Cutoff

5x Cutoff

10x Cutoff

6-MAM 100 101 99.6 97.7 101 99.6 101

Hydroxyalprazolam 99.4 102 96.9 103 99.9 101 98.7

Alprazolam 98.0 101 102 99.5 103 101 95.0

Amphetamine 97.5 100 101 103 103 99.4 94.9

Benzoylecgonine 98.3 99.1 102 103 103 100 94.7

Buprenorphine 86.5 111 112 113 107 89.0 102

Carisoprodol 97.8 97.3 104 102 100 108 93.8

Clonazepam 99.6 102 99.6 98.8 99.8 100 100

Codeine 97.8 100 104 101 99.6 100 98.1

Diazepam 99.1 101 101 98.5 102 101 97.4

EDDP 97.3 99.1 105 101 102 101 94.1

Fentanyl 101 101 101 94.6 97.0 98.9 107

Flunitrazepam 101 101 98.7 97.7 102 102 98.2

Flurazepam 96.6 101 103 103 106 99.7 92.1

Hydrocodone 100 98.0 101 100 101 99.3 100

Hydromorphone 101 99.8 97.7 99.6 100 102 99.7

Lorazepam 98.9 105 98.0 98.1 98.7 101 101

MDA 98.9 100 101 101 101 99.8 98.3

MDEA 99.6 100 98.9 101 103 101 95.7

MDMA 99.9 100 99.5 99.3 103 100 97.3

Meperidine 95.7 101 104 104 103 98.2 94.2

Meprobamate 97.0 100 98.6 106 99.1 95.2 103

Methadone 99.6 101 99.7 101 97.8 102 99.9

Methamphetamine 95.6 99.8 104 105 105 98.9 91.6

Midazolam 96.8 103 102 99.6 103 101 95.4

Morphine 99.6 102 98.9 98.3 101 101 99.5

Naloxone 101 98.4 102 97.5 98.2 100 103

Naltrexone 96.3 104 104 98.8 100 100 97.3

Norbuprenorphine 101 105 96.5 96.6 99.9 101 99.9

Nordiazepam 103 97.6 99.2 97.7 99.9 101 101

Norfentanyl 103 99.3 101 97.4 99.2 101 99.9

Normeperidine 100 100 99.6 99.3 99.4 99.6 101

Norpropoxyphene 99.3 99.5 103 98.9 99.6 99.9 99.9

Oxazepam 100 100 100 98.9 100 101 99.4

Oxycodone 98.1 101 102 100 100 99.4 98.5

Oxymorphone 102 99.3 99.8 96.5 102 101 100

PCP 105 94.9 99.4 101 95.0 101 103

Propoxyphene 101 113 101 88.4 97.5 96.5 107

Sufentanil 92.7 98.5 103 104 102 98.1 100

Temazepam 98.9 100 101 100 99.5 100 99.0

Tramadol 103 99.2 97.9 97.1 99.5 101 103

Analyte 40 % Cutoff

60 % Cutoff

75 % Cutoff

Cutoff 3x Cutoff

5x Cutoff

10x Cutoff

6-MAM 7.40 7.51 9.91 7.56 6.20 8.30 5.36

Hydroxyalprazolam 8.95 6.78 11.4 6.61 5.92 6.17 7.23

Alrpazolam 3.51 5.53 7.26 4.17 4.39 4.93 3.17

Amphetamine 2.43 4.79 3.50 2.43 1.87 1.39 2.72

Benzoylecgonine 3.38 2.47 1.97 2.69 3.61 2.37 1.82

Buprenorphine 14.0 12.3 7.88 9.36 4.84 3.89 2.62

Carisprodol 15.2 8.10 5.34 11.9 7.43 14.4 12.0

Clonazepam 7.69 6.33 9.06 3.90 3.52 2.18 3.81

Codeine 5.45 6.23 4.01 6.10 3.70 4.94 5.08

Diazepam 2.57 7.41 4.74 3.02 5.71 4.00 2.68

EDDP 5.37 6.59 4.16 6.43 3.98 2.89 3.71

Fentanyl 5.14 3.19 5.76 6.80 3.81 2.39 2.27

Flunitrazepam 5.67 6.28 6.01 3.95 7.59 4.30 2.09

Flurazepam 7.43 3.11 5.86 4.21 2.40 3.94 3.84

Hydrocodone 4.05 3.60 4.87 3.48 7.03 1.85 2.75

Hydromorphone 6.20 3.73 3.70 5.30 2.48 1.88 2.86

Lorazepam 10.6 5.92 8.16 7.09 9.90 4.45 5.68

MDA 10.2 8.22 7.73 7.69 7.51 6.92 4.49

MDEA 9.15 5.21 3.38 3.36 2.26 2.62 2.14

MDMA 9.01 6.15 2.58 3.18 4.11 3.13 3.27

Meperidine 2.43 1.47 1.86 1.60 1.73 2.33 2.07

Meprobamate 11.2 12.2 6.44 11.5 11.7 13.0 13.7

Methadone 3.01 3.00 3.04 3.50 4.47 2.51 3.30

Methamphetamine 3.12 3.53 2.36 1.47 2.58 2.08 1.96

Midazolam 4.51 7.19 7.70 5.70 4.81 3.86 4.24

Morphine 5.86 3.88 4.42 4.65 2.48 2.24 2.49

Naloxone 7.87 4.61 8.06 2.89 5.00 5.03 4.70

Naltrexone 10.5 6.75 6.80 5.81 4.19 6.58 4.13

Norbuprenorphine 6.31 7.20 5.96 7.29 2.17 2.48 1.85

Nordiazepam 4.27 4.37 2.83 6.06 1.88 3.87 3.86

Norfentanyl 6.41 3.50 4.31 3.76 2.52 2.11 2.33

Normeperidine 4.06 4.05 2.57 2.10 3.45 1.89 2.66

Norpropoxyphene 3.01 4.44 1.51 5.74 3.11 5.11 3.92

Oxazepam 11.5 6.55 8.93 8.09 5.86 6.00 7.09

Oxycodone 7.57 7.09 5.89 5.68 5.87 8.10 6.70

Oxymorphone 6.28 4.93 7.22 6.19 5.12 6.27 6.06

PCP 11.0 9.44 6.97 13.8 13.9 4.90 11.0

Propoxyphene 13.8 1.24 11.3 7.94 10.8 14.2 7.78

Sufentanil 8.45 2.80 7.39 7.36 5.27 5.63 4.03

Temazepam 6.23 3.66 4.91 2.39 2.45 3.64 4.84

Tramadol 2.10 3.87 1.88 1.31 3.22 2.04 2.57

APPLICATIONSTN-1141

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Ordering Information

Kinetex HPLC/UHPLC Column Ordering Information

* Kit includes Kinetex® 2.6 µm C18 HPLC/UHPLC column, SecurityGuard™ ULTRA cartridges and holder, in-line filter, Phenex™ Syringe Filters, Verex™ Vial kit, Sure-Lok™ UHPLC Nut and tightening tool, Sure-Lok Fingertight Nut and wrenches.

Complete iMethod™ Kit*Description Part No.iMethod Test for the Full Pain Panel V1.2 KH0-9162

5 µm Columns (mm)

SecurityGuard™ ULTRA Cartridges*

SecurityGuard ULTRA Cartridges*

50 x 2.1 3/pk 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pkPhenyl-Hexyl 00B-4603-AN AJ0-8788 00B-4603-E0 00D-4603-E0 00F-4603-E0 00G-4603-E0 AJ0-8774

for 2.1 mm ID for 4.6 mm ID* SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000.

2.6 μm Analytical Columns (mm)SecurityGuard

ULTRA Cartridges‡

Phases 50 x 4.6 100 x 4.6 150 x 4.6 3/pkPhenyl-Hexyl 00B-4495-E0 00D-4495-E0 00F-4495-E0 AJ0-8774

for 4.6 mm ID

1.7.µm Minibore Columns (mm)7 μm Minibore Columns (m)

SecurityGuard ULTRA Cartridges‡

Phases 50 x 2.1 100 x 2.1 150 x 2.1 3/pkPhenyl-Hexyl 00B-4500-AN 00D-4500-AN 00F-4500-AN AJ0-8788

for 2.1 mm ID

2.6 μm Minibore Columns (mm)SecurityGuard

ULTRA Cartridges‡

Phases 50 x 2.1 100 x 2.1 3/pkPhenyl-Hexyl 00B-4495-AN 00D-4495-AN AJ0-8788

for 2.1 mm ID

‡SecurityGuard ULTRA cartridges require holder, Part No.: AJ0-9000

For AB SCIEX mass spectrometer users, this method can be instantly implemented by installing the iMethod™ Test. iMethod Tests are verified across several laboratories and

contain everything you will need to start running samples including sample preparation recommendations, consumables, LC running conditions, optimized MRM parameters, reporting templates, and complete method documentation.

Visit www.phenomenex.com/iMethod for more information about available iMethod Tests.

If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and comparable dimensions, return the product with comparative data within 45 days for a FULL REFUND.

APPLICATIONSTN-1141

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TN97

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