Skip to Content
Merck
  • Determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (LPME) and gas chromatography-mass spectrometry (GC-MS).

Determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (LPME) and gas chromatography-mass spectrometry (GC-MS).

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2014-12-03)
André Valle de Bairros, Rafael Menck de Almeida, Lorena Pantaleão, Thiago Barcellos, Sidnei Moura e Silva, Mauricio Yonamine
ABSTRACT

In this study, it is shown a method for the determination of benzodiazepines and their main metabolites in urine samples by hollow-fiber liquid-phase microextraction (LPME) in the three-phase mode. Initially, the hydrolysis step was performed using 100 μL of sodium acetate 2.0 mol/L buffer solution (pH 4.5), 25 μL of β-glucuronidase enzyme and incubation for 90 min at 55 °C. In parallel with hydrolysis, the LPME fiber (9 cm) was prepared. Its pores were filled with a mixture of dihexyl ether: 1-nonanol (9:1). Afterwards, a solution of 3.0 mol/L of HCl was introduced into the lumen of the fiber (acceptor phase). After hydrolysis, the fiber was submersed in the alkalinized urine (pH 10) containing 10% NaCl. Samples were then submitted to orbital shaking (2400 rpm) for 90 min. The acceptor phase was later withdrawn from the fiber, dried and the residue derivatized with trifluoroacetic anhydride (TFAA) for 10 min at 60 °C with further addition of N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide containing 1% tert-butyldimethylchlorosilane (MTBSTFA) for 45 min at 90 °C followed by determination by gas chromatography-mass spectrometry (GC-MS). The calibration curves obtained showed linearity over the specified range, with a similar sensitivity to traditional techniques and a higher detection capability compared to most of the miniaturized methods described in the literature. The method has been developed and successfully validated and applied to urine samples from real cases of benzodiazepines intake.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-Butyldimethylchlorosilane, ≥95%
Sigma-Aldrich
tert-Butylchlorodimethylsilane solution, 50 wt. % in toluene
Sigma-Aldrich
Sodium hydroxide solution, BioUltra, for molecular biology, 10 M in H2O
Sigma-Aldrich
1-Octanol, natural, ≥98%, FCC
Supelco
Trifluoroacetic anhydride, for GC derivatization, LiChropur, ≥99.0% (GC)
Supelco
Hydrogen chloride – ethanol solution, ~1.25 M HCl, for GC derivatization, LiChropur
Sigma-Aldrich
3-Ethyl-2,4-pentanedione, mixture of tautomers, 98%
Supelco
Hydrochloric acid solution, volumetric, 0.1 M HCl (0.1N), endotoxin free
Sigma-Aldrich
Hydrogen chloride solution, 3 M in cyclopentyl methyl ether (CPME)
USP
Sodium acetate, United States Pharmacopeia (USP) Reference Standard
Supelco
N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-Butyldimethylchlorosilane, for GC derivatization, LiChropur, ≥95.0% (GC)
Sigma-Aldrich
Sodium hydroxide, beads, 16-60 mesh, reagent grade, 97%
Sigma-Aldrich
Hydrogen chloride solution, 1.0 M in acetic acid
Sigma-Aldrich
Sodium hydroxide, puriss. p.a., ACS reagent, reag. Ph. Eur., K ≤0.02%, ≥98%, pellets
Sigma-Aldrich
Hydrogen chloride solution, 1.0 M in diethyl ether
Sigma-Aldrich
Zirconyl chloride octahydrate, reagent grade, 98%
Sigma-Aldrich
Hydrochloric acid, ACS reagent, 37%
Sigma-Aldrich
Hydrochloric acid, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., fuming, ≥37%, APHA: ≤10
Sigma-Aldrich
Hydrochloric acid, 37 wt. % in H2O, 99.999% trace metals basis
Sigma-Aldrich
Hydrogen chloride solution, 2.0 M in diethyl ether
Sigma-Aldrich
Sodium hydroxide, reagent grade, 97%, powder
Sigma-Aldrich
Hydrochloric acid, meets analytical specification of Ph. Eur., BP, NF, fuming, 36.5-38%
Sigma-Aldrich
Sodium hydroxide solution, purum, ≥32%
Sigma-Aldrich
Hydrochloric acid, puriss., 24.5-26.0%
Sigma-Aldrich
Sodium hydroxide, puriss. p.a., ACS reagent, K ≤0.02%, ≥98.0% (T), pellets
Sigma-Aldrich
Sodium hydroxide, anhydrous, free-flowing, Redi-Dri, reagent grade, ≥98%, pellets
Sigma-Aldrich
Sodium acetate, anhydrous, for molecular biology, ≥99%
Sigma-Aldrich
Sodium acetate, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99.0%
Sigma-Aldrich
Sodium acetate, puriss. p.a., ACS reagent, reag. Ph. Eur., anhydrous
Sigma-Aldrich
Sodium acetate, anhydrous, BioUltra, for luminescence, for molecular biology, ≥99.0% (NT)