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Pain panel extracted from human urine at cutoff - Kinetex C18 2.6µm 50x2.1mm LCMSMS

Sample Preparation Details

Part No:8B-S029-TBJ
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 30 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Urine samples were first hydrolyzed to convert the glucuronide metabolites to native form using ß-glucuronidase. This was done by first weighing 1 g of ß-glucuronidase and diluting with 20 mL of DI water. A 100 µL aliquot of this solution was added to 1 mL of urine and vortexed to mix (6200 units in 1 mL of urine). Samples were incubated for 2 hours at 60 °C, and then centrifuged for 10-15 minutes at 9,500-13,000 rpm to form a pellet of protein and solids. The clarified supernatant (~1 mL) was then removed and diluted with 1 mL of 100 mM ammonium acetate buffer (pH 5.95).

Dry:5 mins under full vacuum
Final Prep and Analysis: Inject: 5 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Morphine 300 
2 Morphine-d3 100 
3 Oxymorphone 300 
4 Hydromorphone 300 
5 Hydromorphone-d6 100 
6 Codeine 300 
7 Codeine-d6 100 
8 Naloxone 300 
9 Oxycodone 300 
10 Oxycodone-d6 100 
11 Naltrexone 300 
12 Hydrocodone 300 
13 cis-Tramadol 300 
14 C13-D3-tramadol 100 
15 Meperidine 300 
16 Meperidine-d4 100 
17 Fentanyl
18 Buprenorphine 300 
19 Buprenorphine-d4 100 
20 Methadone 300 
21 Methadone-d3 100 

Application Detail (App ID: 18850)

Part No:00B-4462-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% Formic acid in H2O
Elution B:0.1% Formic acid in ACN
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 95 5
2 1.3 40 60
3 1.31 5 95
4 1.51 5 95
5 1.52 95 5
6 2.2 95 5

Flow Rate:1.2 mL/min
Col. Temp: 25 °C
Detection: Mass Spectrometer (MS) @ amu (22°C)
Note:ESI conditions
positive mode
QTrap 4000
NEB: 10.00
CUR: 11.00
IS: 4500.00
TEM: 450.00
CAD: 11.00
EP 10.00

Analyte Details

Analyte Detail not available

1
Morphine
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2
Morphine-d3
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3
Oxymorphone
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4
Hydromorphone
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5
Hydromorphone-d6
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6
Codeine
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7
Codeine-d6
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8
Naloxone
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9
Oxycodone
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10
Oxycodone-d6
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11
Naltrexone
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12
Hydrocodone
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13
cis-Tramadol
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14
C13-D3-tramadol

15
Meperidine
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16
Meperidine-d4
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17
Fentanyl
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18
Buprenorphine
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19
Buprenorphine-d4
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20
Methadone
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21
Methadone-d3
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