The analyses of 1,4-Benzodiazepines using the Cogent Diol Column from Urine Samples after Solid Phase Extraction using two types of Retention Modes are shown in this Note.

Four Compounds were well retained and separated in both HPLC Modes shown in Figures A and B; however, the Retention Order was changed depending on the Gradient used. It is worth noticing that the peak intensities were three times higher when using Gradient “A” (ANP mode) compared to Gradient “B” (RP mode).

1. Temazepam 301.0739 m/z [M+H]+
2. Diazepam 285.0790 [M+H]+
3. Nordiazepam 271.0633 [M+H]+
4. Midazolam 326.0855 [M+H]+

Method Conditions
Column: Cogent Diol™, 4.4µm, 1 00Å
Catalog No.: 40060-05P-2
Dimensions: 2.1 x 50 mm
Solvents: A: DI H20 / 0 .1% formic Acid (v/v)   B: Acetonitrile/ 0.1% formic Acid (v/v)

Figure A Figure B
time/(min) %B time/(min) %B
0 85 0 10
6 70 6 10
7 20 7 50
9 20 9 50
10 85 10 10

Post time: 3 min
Injection vol.: 1 µL
Flow rate: 0.4 ml/min
Detection: ESI – POS – Perkin Elmer AxION 2 TOF mass spectrometer
Flow rate: 0.4 ml/min

Samples: Extraction method: Spiked urine sample was loaded into SPE cartridge I (Clean Screen Xcel™ purchased from UCT Bristol, PA, USA) and eluted with 0.78 mL of acetonitrile, 200 microL of 2-propanol, 20 microL of ammonia. After the elution, the sample was dried under N2 gas and dissolved in 100 microL of 50% methanol / 50% DI H2O / 0.1% formic acid.

Before injection, the 10 ppm spiked sample was filtered through a 0 .45µm nylon syringe filter (MicroSolv Tech Corp).

Notes: Benzodiazepines are prescribed for conditions such as anxiety disorders, insomnia, seizures, and alcohol withdrawal. However, they also have potential for abuse as recreational drugs.



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