Multi-residue detection of benzodiazepines by ELISA based on class selective antibodies

Figures & data

Figure 1.  General structure of benzodiazepines.

Table 1. Chemical structures of the benzodiazepines studied.

Benzodiazepines	R1	R2	R3	R5	R7
Nitrazepam	H	=O	H	Phenyl	NO2
Clonazepam	H	=O	H	2-Cl-Phenyl	NO2
7-aminonitrazepam	H	=O	H	Phenyl	NH2
7-aminoclonazepam	H	=O	H	2-Cl-Phenyl	NH2
Delorazepam	H	=O	H	2-Cl-Phenyl	Cl
Chlordiazepoxide	H	NHCH3	H	Phenyl	Cl
Nordazepam	H	=O	H	Phenyl	Cl
Oxazepam	H	=O	OH	Phenyl	Cl
Diazepam	CH3	=O	H	Phenyl	Cl

Figure 2.  Structures of nitrazepam, clonazepam and 7-aminonitrazepam, and 7-aminoclonazepam.

Figure 3.  LC-MS chromatograms of identification of 7-aminonitrazepam.

Figure 4.  LC-MS chromatograms of identification of 7-aminoclonazepam.

Figure 5.  Determination of conjugation density of 7-aminonitrazepam to the carrier protein by UV spectrometry.

Figure 6.  Determination of conjugation density of 7-aminoclonazepam to the carrier protein by UV spectrometry.

Figure 7.  Standard curve of IC-ELISA for nitrazepam determination.

Figure 8.  Standard curve of IC-ELISA for clonazepam determination.

Table 2. Precision to two benzodiazepines using indirect competitive ELISA format.

Benzodiazepines	Linear equation	R ^2	Linear range (µg/l)	IC50 (µg/l)	LOD (µg/l)
Nitrazepam	y = –34.9901log(x) + 64.208	0.9823	0.1500–15.000	2.5468	0.1832
Clonazepam	y = –33.070log(x) + 67.003	0.9853	0.1500–15.000	3.2658	0.2016

Figure 9.  Standard curve of IC-ELISA for benzodiazepines.

Table 3. Cross-reactivities of benzodiazepines and similar substances.

	Cross-reactivity (%)
Compounds	Ab of a-BSA	Ab of b-BSA
Nitrazepam	100	90
Clonazepam	80	100
7-aminonitrazepam	130	80
7-aminoclonazepam	90	105
Delorazepam	80	95
Chlordiazepoxide	15	5
Nordazepam	55	10
Oxazepam	0.5	0.3
Diazepam	0.3	0.3
Triazolam	<0.1	<0.1
Promethazine hydrochloride	<0.1	<0.1

Table 4. Determination of nitrazepam in spiked urine samples by IC-ELISA.

Spiked concentration (µg/l)	Mean±SD (µg/l)	Recovery (%)	CV (%)
5.0	4.1±0.2	82±4	10.7
10.0	9.6±0.7	96±7	13.2
20.0	20.6±1.1	103±5	11.2