Standards and Samples: The standards and samples were prepared as follows:
A certified eight component mix in acetonitrile (250 mg/mL per component) was used (Benzodiazepine mix [Cerilliant, B-033])
and contained the following compounds: alprazolam; clonazepam; diazepam; flunitrazepam; lorazepam; nitrazepam; oxazepam; and
Working standards for instrument calibration were prepared in 20:80, acetonitrile:H2O solution over a range of 1–500 ng/mL.
Samples were prepared by spiking preserved synthetic urine (Surine, Cerilliant, 720-1) with the benzodiazepine mix at concentrations
of 10 ng/mL (low spike) and 150 ng/mL (high spike) of each component.
UHPLC Method: The instruments used to conduct the analysis included a PerkinElmer Flexar FX-10 binary pump, Flexar FX-UHPLC autosampler,
and Flexar Peltier column oven. The column used was a 100 mm × 2.1 mm, 2.7-µm SPP Phenyl-Hexyl (N9308485) (PerkinElmer). Mobile
phase A: LC–MS-grade water with 0.1% formic acid and 0.5% acetonitrile. Mobile phase B: LC–MS-grade acetonitrile with 0.1%
formic acid. Column temperature: 35 °C
MS Method: The spectrometer used in this analysis was a Flexar SQ300 single-quad mass spectrometer (PerkinElmer) set to the following
specifications: Drying gas temperature: 325 °C; drying gas (N2) flow rate: 15 L/min; positive ESI mode with capillary exit voltage for all analytes was set at 100 V and dwell time for
all ions was 25 ms; single ion monitoring (SIM) was used for all eight compounds.
Table 1: Gradient programme.
Calibration Results: The calibration standards used in this experiment were: 1, 5, 10, 50, 100, 250, and 500 ng/mL.
The limit of quantitation (LOQ), or concentration at which quantitative results can be reported with a high degree of
confidence, was equal to or below 1 ng/mL
All benzodiazepines analyzed showed excellent curve fits with r2 values of 0.999, demonstrating that the assay was valid over the clinically relevant range.
SPE Method: Spiked Surine samples were prepared at concentrations of 10 ng/mL and 150 ng/mL. These levels were chosen to demonstrate
precision and accuracy of the method at both low and high benzodiazepine concentrations. Extractions were conducted on Supra-Poly
(PerkinElmer) 30 µm and 60 µm HLB 96-well plates, 30 mg × 2 mL (N9306698 & N9306695, respectively).
The extraction procedure for the benzodiazepines was as follows: The 96-well extraction plates were first conditioned with
1 × 1 mL methanol followed by 2 × 1 mL H2O. A 1 mL spiked sample of Surine was loaded onto the 96-well plates. The sample was then washed with 2 × 1 mL H2O, 1 × 1 mL 20% acetonitrile and dried for 5–10 min under vacuum. The sample was eluted from the 96-well plate with 1 mL of
acetonitrile. The sample was dried under N2 at <40 °C and redissolved in 500 µL of 20% acetonitrile in H2O.
Extraction Results: Extraction results are provided for both 10 ng/mL spiked Surine and 150 ng/mL spiked surine samples (Table 2). Extractions
were conducted on two solid-phase media "A" and "B", differing only in the particle sizes (30-µm and 60-µm particle diameter,
respectively) to assess the two media types for recovery and reproducibility for the benzodiazepine mixture at a low and a
high concentration spike.
Table 2: The extraction results for 10 ng/mL spiked Surine samples (low spike) and results for 150 ng/mL spiked Surine samples
(high spike) extracted on both solid-phase media A and B.
Even with small detection windows, using SPE with LC–MS offers a highly reliable method to analyze benzodiazepines in urine
with a recovery rate that exceeds 90%. It provides clean extracts and a single, simple protocol for a wide range of compounds.
SPE also allows for robust and reproducible results even when the sample matrices vary from patient to patient. Finally, using
the SPE process to test for benzodiazepines in urine can be automated, which speeds up procedures and helps laboratories to
become more efficient and productive.
Jason P. Weisenseel is currently the Chromatography Technical Leader for Aftermarkets with PerkinElmer, Environmental Health, Inc. (Shelton, Connecticut,
USA), and a visiting research associate at Vanderbilt University (Nashville, Tennessee, USA). He was a tenured assistant professor
of Chemistry at the University of North Alabama (Florence, Alabama, USA) prior to joining PerkinElmer, where he taught chemical
instrumental analysis. Weisenseel earned a PhD in chemistry from Vanderbilt University in 2000.