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Benzodiazepines from urine using Strata-X-Drug N on a Kinetex C18 2.6µm, 50x2.1mm

Sample Preparation Details

Part No:8B-S129-ECH
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 100 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Pre-Treatment: To 2mL urine, add 1mL beta-glucuronidase (contains 5,000 F units/mL Patella vulgata in 100mM acetate buffer(pH 5.0).
Mix/vortex.
Hydrolyze for 3 hours at 60C.
Let cool and add 1mL 100mM Phosphate buffer (pH 6.0).
Centrifuge for 5 minutes at 5000 rpm and discard pellet. Load directly onto SPE cartridge - NO CONDITIONING REQUIRED (Extraction automated using a Gilson GX-274 ASPEC liquid handler)
Dry:10 minutes @ full vacuum
Final Prep and Analysis: Evaporate eluant under a stream of N2 gas at 50ºC. Reconstitute with 1mL of 35% methanol Inject: 5 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 alpha-Hydroxyalprazolam 300  92.4 10.6
2 Oxazepam-d5 300  100 0.01
3 Oxazepam 300  105.3 5.6
4 Alprazolam-d5 300  100 0.01
5 Alprazolam 300  98.4 2.1
6 Nordiazepam-d5 300  100 0.01
7 Nordiazepam 300  103.7 3.7
8 Lorazepam-d4 300  100 0.01
9 Lorazepam 300  101.1 1
10 Clonazepam-d4 300  100 0.01
11 Clonazepam 300  99.6 0.5
12 Temazepam-d5 300  100 0.01
13 Temazepam 300  104.6 3.9
14 Diazepam-d5 300  100 0.01
15 Diazepam 300  101.1 1

Application Detail (App ID: 19714)

Part No:00B-4462-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% Formic acid
Elution B:0.1% Formic acid in methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 65 35
2 4 5 95
3 4.01 65 35
4 7 65 35

Flow Rate:300 µL/min
Col. Temp: ambient
Detection: Mass Spectrometer (MS) @ amu (22°C)
Detector Info: AB Sciex API 3000™

Source: Cur-11, CAD-12, IS-4500, Tem-450, NEB-12
Dwell: 25ms
Polarity: Positive
Note:SecurityGuard™ ULTRA Guard Cartridge System extends column lifetime.
- SecurityGuard ULTRA Cartridges, UHPLC C18 for 2.1mm ID Columns, 3/Pk Part No.: AJ0-8782
- Holder Part No.: AJ0-9000

Analyte Details

Analyte Detail not available

1
alpha-Hydroxyalprazolam
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2
Oxazepam-d5
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3
Oxazepam
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4
Alprazolam-d5
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5
Alprazolam
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6
Nordiazepam-d5
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7
Nordiazepam
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8
Lorazepam-d4
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9
Lorazepam
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10
Clonazepam-d4
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11
Clonazepam
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12
Temazepam-d5
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13
Temazepam
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14
Diazepam-d5
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15
Diazepam
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