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HPLC Method for Simultaneous Determination of 6Benzodiazepines

Method: HPLC
Matrix: Medicine
Application No.: 101925
Componds: Clonazepam, Estazolam, Triazolam, Alprazolam, Chlordiazepoxide, Diazepam
Columns: Inspire C18 5 μm 250 x 4.6 mm
Cat No.: 81006
Sample Pretreatment: Standards: Individual stock solutions were prepared in acetonitrile/methanol (1/1)at a concentration of 0.5 mg/mL.
A working solution of the mixture of all 6 drugs was obtained by dilution of the stock solution with
methanol/water (9/1). Each drug concentration in the working solution is listed in Table 1.
All the stock solutions should be stored at -20 ℃. Working solutions are prepared when needed and used
out as soon as possible.
Conditions: Mobile Phase:
A: 100 mM ammonium acetate (pH 4.2):Methanol = 7/3
B: 100 mM ammonium acetate (pH 4.2):Methanol = 5/95
Gradient: hold 2% B for 2.5 min, 2 to 50% B in 4.5 min, 50 to 60% B in 13 min,
hold 60% B for 2 min, 60 to 2% B in 1 min, equilibrate at 2% B for 7 min.
Flow Rate: 1.0 mL/min;
Injection: 10 µL;
Temperature: 300C;
Wavelength: 254nm
Publisher: Dikma Technologies Inc.
Keyword: Clonazepam, Estazolam, Triazolam, Alprazolam, Chlordiazepoxide, Diazepam, HPLC
Abstract: In this application note, a new method for analysis of 6 benzos was developed by using the DIKMA Inspire 5 µm C18 column. All analytes were eluted from the column within 21 mins with good peak shape and resolution.

No.

Compounds

Concentration (µg/mL)

Retention Time(min)

Resolution

Asymmetry

1

Clonazepam

20

13.56

-

1.01

2

Estazolam

20

14.45

3.91

1.02

3

Triazolam

40

14.95

2.04

1.02

4

Alprazolam

20

15.80

3.28

1.03

5

Chlordiazepoxide

10

18.15

7.99

1.04

6

Diazepam

20

20.63

7.51

1.04