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

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Sample Preparation Details

Condition 1 :1 at 1.0

Condition 2 :1 at 1.0

Load :Load 0.20 mL Sample diluted in 0.1% Formic acid/water

Wash 1 :1 mL 0.1% Formic acid/water at 1.0 mL/min

Wash 2 :1 mL 30% methanol at 1.0 mL/min

Dry :3 to 4 mins at high vacuum (10 of Hg)

Elution 1 :0.5 mL 2% ammonium hydroxide/methanol: acetonitrile (1:1) at 1.0 mL/min

Elution 2 :0.5 mL 2% ammonium hydroxide/methanol: acetonitrile (1:1) at 1.0 mL/min

Evaporate: :Evaporate to dryness

Reconstitute :200 µL HPLC mobile phase A/B=85/15

Notes :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.

Application Detail (App ID: 20847)

Column: Kinetex® 2.6 µm Phenyl-Hexyl 100 Å, LC Column 50 x 4.6 mm, Ea

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: 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

Morphine

Morphine-d6

Hydromorphone

Hydromorphone-d6

Naloxone

Codeine

Oxymorphone

Naltrexone

Amphetamine

Amphetamine-d5

6-MAM

Oxycodone

Hydrocodone

Hydrocodone-d6

Methamphetamine

MDA

MDMA

MDEA

Norfentanyl

Tramadol

Benzoylecgonine

Benzoylecgonine-d3

Meperidine

Meperidine-d4

Normeperidine

Normeperidine-d4

Meprobamate

Norbuprenorphine

PCP

PCP-D5

Fentanyl

Fentanyl-d5

EDDP

Flurazepam

Norpropoxyphene

Norpropoxyphene-d5

Sufentanil

Buprenorphine

Propoxyphene

Carisoprodol

Methadone

Methadone-d3

Midazolam

Lorazepam

Clonazepam

Hydroxyalprazolam

Oxazepam

Flunitrazepam

Alprazolam

Temazepam

Nordiazepam

Nordiazepam-d5

Diazepam

6-MAM-d3

Methamphetamine-d5