Benzodiazepine Panel in Urine

Authors: Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Benzodiazepine, Drugs of Abuse, Urine, LDTD-MS/MS
From: Phytronix Technologies, Québec, Canada
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Introduction

Toxicological labs need to screen different drugs classes like benzodiazepines, anxiolytics and anticonvulsants using a generic sample preparation and fast analytical technique. Benzodiazepines are a drug class that is prescribed as a tranquilizer and is often abused in conjunction with opioids and alcohol. Our goal for this application note is to optimize an automated sample preparation method for a benzodiazepine panel in urine using a single operation in LDTD®-MS/MS.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a quick and simple sample preparation. Twenty-one drugs of the benzodiazepine class are analyzed simultaneously with quantitative screening results obtained in less than 8 seconds per sample. Each drug has been screened based on the industry cut-offs requirement.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD technology for mass spectrometry. Luxon Ion Source uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy, and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic saturation characterize this type of ionization and is the result of the absence of solvent and mobile phase.

Figure 1 – Luxon Ion Source®

 

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Sample Collection

Urine samples were collected and transferred into barcoded tubes, readable by the Azeo Liquid Handler.

 

Figure 3 – Automated extraction system – Azeo Liquid Handler

Sample Extraction (SALLE) : Panel 1

Each barcoded vial was scanned by the Azeo Liquid Handler and an automatic batch file was created. The Azeo extraction system (Figure 3) is used to extract the samples using the following conditions:

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:

MS/MS

MS model: QTrap® System 5500, Sciex

Scan Time: 5 msec

Ionization: APCI

Analysis Method: Positive MRM mode

 

Table 1 – MRM transitions for LDTD-MS/MS
Drugs Transition CE
Meprobamate 219.1 → 158.1 10
Carisoprodol-d7 269.2 → 183.2 10
Nordiazepam 271.1 → 140.1 27
7-Aminoflunitrazepam 284.0 → 226.0 50
Diazepam 285.1 → 154.1 32
7-AminoClonazepam 286.1 → 222.2 30
Oxazepam 287.1 → 241.1 30
7-AminoClonazepam-d4 290.1 → 226.0 30
7-Aminoflunitrazepam-d7 291.1 → 230.0 50
Oxazepam-d5 292.1 → 246.1 30
Temazepam 301.1 → 255.1 25
Temazepam-d5 306.1 → 260.1 25
Zaleplon 306.1 → 265.2 20
Zolpidem 308.1 → 236.1 35
Alprazolam 309.1 → 274.1 35
Alprazolam-d5 314.1 → 286.1 35
Clonazepam 316.0 → 214.0 50
Lorazepam 321.0 → 275.0 30
(Alpha) Hydrozyalprazolam 325.1 → 205.1 54
Midazolam 326.1 → 291.1 35
Clozapine 327.1 → 270.1 30
2-Hydroxyethylflurazepam 333.1 → 194.1 30
Alpha-OH-Midazolam 342.1 → 203.1 35
Etizolam 343.1 → 314.0 25
(Alpha) Hydroxytriazolam 359.0 → 331.0 36
Flurazepam 388.1 → 315.0 27

 

Results and Discussion

Initial Cut-Off Test (ng/mL)

A drug list and screening cut-offs required by toxicological labs can be found in Table 2.

Table 2 – Analytes cut-offs
Analyte Cut-off (ng/mL) Analyte Cut-off (ng/mL)
(Alpha) Hydroxyalprazolam 50 Alprazolam 50
7-Aminoclonazepam 50 Clozapine 50
Clonazepam 50 Etizolam 50
Diazepam 50 Flurazepam 50
Midazolam 50 Lorazepam 50
(Alpha) OH-Triazolam 50 Meprobamate 100
2-Hydroxyethylflurazepam 50 Nordiazepam 50
Zaleplon 50 Oxazepam 50
7-Aminoflunitrazepam 50 Temazepam 50
Alpha-OH-Midazolam 50 Zolpidem 50

 

Precision / Accuracy

Spiked samples around the decision point (50% cut-off: QC-L, cut-off: CO and 200% cut-off: QC-H) and blank solutions are used to validate the precision of the method. The peak area against the internal standard (IS) ratio was used to normalize the signal. Replicate extractions are deposited onto a LazWell™ plate and dried before analysis.

The following acceptance criteria were used:

For the inter-run precision experiment, each fortified sample set is analyzed in triplicate on five different days. Table 3 shows the inter-run precision results. No overlapping at the cut-off is observed for 2-Hydroxyethylflurazepam and the %CV was below 20%. Similar results are obtained for the other drugs in the panel.

 

Table 3 – Inter-Run Precision
2-Hydroxyethylflurazepam QC-L CO QC-H
Conc (ng/mL) 25 50 100
N 15 15 15
Mean (ng/mL) 22.9 51.2 103.9
SD 1.6 1.7 3.1
%CV 7.2 3.3 3.0
Mean – 2SD (ng/mL) 19.7 47.8 97.7
Mean + 2SD (ng/mL) 26.3 54.6 110.1

For the intra-run precision experiment, each fortified sample is extracted and analyzed in 8 replicates. Area ratio results are plotted using the ± 2 STD error bars. Figure 4 shows the intra-run results for 2-Hydroxyethylflurazepam. No overlapping is observed for each concentration and the %CV was below 20%. Similar results are obtained for the other drugs in the panel.

Precission of Intra-Run Assay of 2-Hydroxyethulflurazepam
Figure 4 – Intra-Run Precision Curves for 2-Hydroxyethylflurazepam

 

Multi-Matrix Validation

Urine samples were collected from 63 patients. Samples were analyzed using LC-MS/MS reference method and LDTD-MS/MS method. The method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy are verified as followed:

 

LC-MS/MS

Yes No
LDTD-MS/MS Yes TP (True positive) FP (False positive)
No FN (False negative) TN (True negative)

Where:

Sensitivity: (TP / (TP + FN))

Specificity: (TN / (TN + FP))

PPV: (TP / (TP + FP))

NPV: (TN / (TN + FN))

Accuracy: ((TP+TN) / (TP + FN+TN+FP))

 

Table 4 shows the analysis results of 63 real samples for 2-Hydroxyethylflurazepam.

Table 4 – 2-Hydroxyethylflurazepam results
 
 
LC-MS/MS
Yes No
LDTD-MS/MS Yes TP=8 FP=0
No FN=0 TN=55

 

Validation results are reported in Table 5 for 2-Hydroxyethylflurazepam. Similar results are obtained for the other drugs.

Table 5 – Validation results for 2-Hydroxyethylflurazepam
Parameters 2-Hydroxyethylflurazepam
Sensitivity (%) 100
Specificity (%) 100
PPV (%) 100
NPV (%) 100
Accuracy (%) 100

Conclusion

Luxon Ion Source® combined to a Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of a benzodiazepine panel in urine using a simple and automated sample preparation method.