Table VII.
Analytical Methods Applied to the Analysis of Benzodiazepines in Biological Matrices from Alleged DFSA Samples
| Target analytes | Biological matrix |
Sample pretreatment | Analyte extraction | Recovery of BZDs [%] | Analytical method | Limits of detection | Reference |
|---|---|---|---|---|---|---|---|
| 18 BZDs (1 metabolite) and other drugs Total: 46 compounds |
Whole blood | Adjust to pH 4 (ammonium acetate buffer) |
SPE Sorbent: Oasis® MCX Elution: ethyl acetate (5% ammonium hydroxide) |
17–135 | UHPLC–TOF-MS | LOQ: 0.1–3 ng/g | (20) |
| 24 BZDs (7 metabolites) and other drugs Total: 65 compounds |
Whole blood | Deproteinization with methanol Adjust to pH 9 (saturated carbonate buffer) |
DLLME Extractant: Chloroform Disperser: Methanol with addition of sodium chloride |
Mean: 8– 69 | UHPLC–MS-MS Acquisition: SRM |
LOD: 2 ng/mL LOQ: 5 ng/mL except for alprazolam and chlordiazepoxide |
(85) |
| 23 BZDs (2 metabolites)
and other drugs Total: 96 compounds |
Whole blood | PP and PLR Protein precipitation with acetonitrile/methanol (95:5, v/v), supernatant: formic acid 1% in acetonitrile (v/v) on Phree® Phospholipid Removal Plates |
Mean: 69 for all drugs | HPLC–MS-MS Acquisition: SRM |
LOD: 0.1–10 ng/mL | (86) | |
| 18 BZDs (1 metabolite) and other drugs Total: 128 compounds |
Urine | Hydrolysis with β-glucuronidases/arylsulfatase at pH 5.5 (acetate buffer) at 55℃ for 1 h | SPE Sorbent: Oasis® HLB Elution: methanol and methanol/isopropanol (3:1, v/v) |
16–108 | GC--EI-MS Derivatization: BSTFA + 1% TCMS in ethyl acetate/acetonitrile (1:1, v/v) |
LOD: 120–12,000 ng/mL LOQ: 200- 20 000 ng/mL |
(87) |
| 21 BZDs (4 metabolites)
and Z-drugs Total: 23 compounds |
Urine | Hydrolysis with β-glucuronidase at pH 6.0 at 55℃ for 1 h, then alkalization at pH 7.5 with phosphate buffer | LLE Dichloromethane/propan-2-ol mixture (85:15, v/v) |
Mean: 55.5–98.3 | HPLC–MS-MS Acquisition: SRM |
LOD: 0.5–30 ng/mL LOQ: 2–100 ng/mL |
(88) |
| 5 BZDs (2 metabolites) | Urine | Adjust to pH 9.5 (carbonate buffer) |
LLE Ethyl acetate |
70.5– 96.7 | HPLC–MS-MS Acquisition: MRM |
LOD: 0.125–1 ng/mL LOQ: 0.25–5 ng/mL |
(89) |
| 22 BZDS (4 metabolites) and other drugs Total: 91 compounds |
Urine | Both hydrolyzed and nonhydrolyzed samples Hydrolysis with β-glucuronidase at 55℃ for 30 min | Directly (without extraction) | Mean: 95 for all drugs | HPLC–MS-MS Acquisition: MRM |
N/S | (90) |
| 24 BZDs (5 metabolites) and other drugs Total: 54 compounds |
Urine | Adjust to pH 9.5 (sodium bicarbonate buffer) |
LLE Ethyl acetate |
89.6–110.3 | HPLC–MS-MS Acquisition: MRM |
LOD: 0.5–5 ng/mL LOQ: 1–10 ng/mL |
(24) (Methods in (91)) |
| 18 BZDs (6 metabolites) | Hair | Segmentation and rinsing twice with dichloromethane. Drying and segments cut into pieces <3 mm and pulverized in phosphate buffer (pH 8.4 at RT for 1 h) |
LLE Dichloromethane |
39.4–102.6 | HPLC–MS-MS Acquisition: MRM |
LOD: 0.0005–0.002 ng/mg | (28) |
| 18 BZDs (2 metabolites) and other drugs Total: 35 compounds |
Hair | Decontamination with two washes with water followed by two washes with dichloromethane. Drying and segmentation into <1 mm. Incubation overnight at RT with phosphate buffer pH 5. |
LLE Dichloromethane/ether (70:30, v/v). |
N/S | HPLC–MS-MS Acquisition: MRM |
LOD: 0.0005–0.01 ng/mg LOQ: 0.0005–0.01 ng/mg |
(92) |
| 13 BZDs (3 metabolites) and other drugs Total: 52 compounds |
Hair | 10 mg hair, one wash with isopropanol and two with H2O. Drying and extraction. Segmentation in 1–2 mm segments or pulverization. Incubation overnight at 37℃ |
Extraction Methanol:acetonitrile:ammonium formate (pH 5.3) Filtration PTFE filter |
87–102 | UHPLC–TOF-MS | LOD: 0.01–0.04 ng/mg LOQ: 0.05 ng/mg |
(29) (Methods in (93) |
| 20 BZDs (6 metabolites)
and 3 Z-drugs Total: 23 compounds |
Hair | Decontamination with H2O, acetone and hexane. Pulverization in a bench-top mill. |
Extraction under shaking 1st step: methanol 2nd step: methanol/ammonium formate buffer (1:1, v/v) at pH 3.5 |
N/S | UHPLC–MS-MS Acquisition: MRM |
LOQ: 0.0005–0.01 ng/mg | (94) |
| 28 BZDs (9 metabolites) and other drugs Total: 200 compounds |
Blood | Deproteinization with methanol, adjust to pH 9 (saturated carbonate buffer) | DLLME Extractant: chloroform Disperser: methanol with addition of sodium chloride |
Mean: 8.0–69.3 | UHPLC–MS-MS Acquisition: MRM |
LOD: 2 ng/mL LOQ: 5 ng/mL except for alprazolam and chlordiazepoxide |
(95) (Methods for blood in (85) |
| Urine | Hydrolysis with β-glucuronidase in acidic medium | Dilution and shoot Aqueous formic acid |
N/S | UHPLC–MS-MS Acquisition: MRM |
LOD: 1–5 ng/mL (not determined for hydroxymidazolam) |
||
| Hair | Decontamination with two washes with aqueous tween 80 and one wash with acetone. Segmentation into 1 mm. | Extraction Aqueous formic acid at 40℃ overnight |
N/S | UHPLC–MS-MS Acquisition: MRM |
N/S |
DLLME: dispersive liquid–liquid microextraction; LLE: liquid–liquid extraction; LOD: limit of detection; LOQ: limit of quantitation; PP: protein precipitation; RT: room temperature; SRM: selected reaction monitoring.