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Applications

Pain Panel using Strata-X-Drug B and Kinetex Phenyl-Hexyl 2.6u 50x4.6mm

Sample Preparation Details

Part No:8B-S128-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:Sample Hydrolysis: Measure 200uL Urine (calibrator, control) in a 1.5mL micro centrifuge tube, add 20 uL combined IS (Internal Standard) spiking solution.
Vortex 10-15 secs followed by adding 100uL 0.1M pH 4.0 ammonium acetate buffer to each tube, mix/vortex another 15 secs.
Add 40uL
beta-Glucoronidase solution (100,000 units/mL).
Vortex 10-15 secs.
Incubate for 2 hours in a shaker at 55o C to complete hydrolysis of the glucuronides.
Add 400ul of 0.1% FA (formic acid) followed by vortexing for another 60 secs.
Centrifuge the hydrolyzed samples at appx 21000 g for 10 mins (to separate any proteins). Collect Supernatant
Dry:3 to 4 mins at high vacuum (10" of Hg)
Final Prep and Analysis: Load: Samples from hydrolysis Using b-Glucoronidase as below
To 200 µL Urine (calibrator, control) in a 1.5 mL micro centrifuge tube, add 20 µL combined IS (Internal Standards) spiking solution. Vortex 10-15 seconds followed by adding 100 µL 0.1 M Ammonium acetate buffer (pH 4.0) to each tube, mix/vortex another 15 seconds. Add 40 µL ß-Glucoronidase solution (100,000 units/mL). Vortex 10-15 seconds. Incubate for 2 hours in a shaker at 55 °C to complete hydrolysis of the glucuronides. Add 400 µl of 0.1% Formic acid and vortex for another 60 seconds. Centrifuge the hydrolyzed samples at 21,000 g for 10 minutes (to separate any proteins). Collect supernatant for further cleanup via Solid Phase Extraction (SPE).
Wash 1: 1 mL 0.1% FA
Wash2: 1 mL 30% methanol
Dry: 3 to 4 mins at high vacuum (~10” of Hg)
Elute: 2x500 uL 2% ammonium hydroxide/methanol: acetonitrile (1:1)
Blow Down: To dryness under nitrogen at 40-45o C
Reconstitute: With 200 uL of 85/15 (A/B) of LC mobile phase
(Note:
1. No conditioning or equilibration needed in extraction)
2.Total of 15 internal standard used for the quantitation of 41 analytes. Inject: 10 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Morphine 25  115
2 Morphine-d6 500 
3 Hydromorphone 25  110
4 Hydromorphone-d6 500 
5 Naloxone 25  103
6 Codeine 25  111
7 Oxymorphone 25  107
8 Naltrexone 25  108
9 Amphetamine 125  99
10 Amphetamine-d5 2500 
11 6-MAM 2.5  99
12 Oxycodone 25  113
13 Hydrocodone 25  113
14 Hydrocodone-d6 312 
15 Methamphetamine 125  113
16 MDA 62.5  99
17 MDMA 62.5  102
18 MDEA 62.5  104
19 Norfentanyl 2.5  112
20 Tramadol 25  96
21 Benzoylecgonine 37.5  100
22 Benzoylecgonine-d3 750 
23 Meperidine 250  95
24 Meperidine-d4 3000 
25 Normeperidine 25  108
26 Normeperidine-d4 500 
27 Meprobamate 50  108
28 Norbuprenorphine 25  93
29 PCP 6.25  120
30 PCP-D5 125 
31 Fentanyl 0.75  110
32 Fentanyl-d5 100 
33 EDDP 25  113
34 Flurazepam 25  117
35 Norpropoxyphene 37.5  103
36 Norpropoxyphene-d5 750 
37 Sufentanil 0.75  84
38 Buprenorphine 25  100
39 Propoxyphene 37.5  98
40 Carisoprodol 50  112
41 Methadone 25  101
42 Methadone-d3 500 
43 Midazolam 25  114
44 Lorazepam 25  102
45 Clonazepam 25  117
46 Hydroxyalprazolam 25  111
47 Oxazepam 25  99
48 Flunitrazepam 25  98
49 Alprazolam 25  101
50 Temazepam 25  104
51 Nordiazepam 25  110
52 Nordiazepam-d5 500 
53 Diazepam 25  114
54 6-MAM-d3 500 
55 Methamphetamine-d5 2500 

Application Detail (App ID: 20847)

Part No:00B-4495-E0
Dimensions:50 x 4.6 mm ID
Elution Type:Gradient
Elution A:10mM ammonium formate
Elution B:0.1% Formic acid/methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 95 5
2 4 0 100
3 5 0 100
4 5.1 95 5
5 7 95 5

Flow Rate:0.6 mL/min
Col. Temp: ambient
Detection: Mass Spectrometer (MS) @ amu (22°C)
Detector Info: AB Sciex API 5000™
Note:MS/MS Parameters
Detection: API 5000 MS/MS, TurboIonspray, positive polarity
CAD Gas: 7; GS1, GS2 = 50; curtain gas: 20, ESI spray voltage: 5500; Temp: 650 deg C

Analyte Details

Analyte Detail not available

1
Morphine
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2
Morphine-d6
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3
Hydromorphone
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4
Hydromorphone-d6
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5
Naloxone
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6
Codeine
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7
Oxymorphone
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8
Naltrexone
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9
Amphetamine
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10
Amphetamine-d5

11
6-MAM
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12
Oxycodone
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13
Hydrocodone
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14
Hydrocodone-d6
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15
Methamphetamine
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16
MDA
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17
MDMA
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18
MDEA
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19
Norfentanyl
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20
Tramadol
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21
Benzoylecgonine
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22
Benzoylecgonine-d3
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23
Meperidine
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24
Meperidine-d4
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25
Normeperidine
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26
Normeperidine-d4
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27
Meprobamate
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28
Norbuprenorphine
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29
PCP
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30
PCP-D5
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31
Fentanyl
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32
Fentanyl-d5
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33
EDDP
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34
Flurazepam
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35
Norpropoxyphene
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36
Norpropoxyphene-d5
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37
Sufentanil
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38
Buprenorphine
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39
Propoxyphene
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40
Carisoprodol
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41
Methadone
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42
Methadone-d3
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43
Midazolam
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44
Lorazepam
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45
Clonazepam
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46
Hydroxyalprazolam
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47
Oxazepam
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48
Flunitrazepam
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49
Alprazolam
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50
Temazepam
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51
Nordiazepam
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52
Nordiazepam-d5
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53
Diazepam
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54
6-MAM-d3
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55
Methamphetamine-d5
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