Benzodiazepine Panel Screening in Hair

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

During the last few decades, research work has been done to investigate the different drugs of abuse in different types of biological samples (urine, saliva, hair, etc). One of the advantages of hair sample is the noninvasive collection.

Our goal for this application note is to develop a sample preparation method for a drug panel in hair using a single operation in LDTD-MS/MS and the Precellys 24 Touch (Bertin Technologies, Montigny-les-Bretonneux, France).

A proper sample preparation protocol is critical for MS-based analysis workflows. Indeed, the quality and reproducibility of the drug extraction can strongly influence MS results. This requires choosing an optimal protocol for the sample preparation step. The 3D-beating technology is considered the gold standard for sample pulverization. For this reason, Bertin Technologies has chosen 3-dimensional bead-beating technology to power the Precellys 24 Touch homogenizer.

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 drugs 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 required.

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

Ten milligram hair samples (approximately 1 cm length) were transferred into the Precellys MK28R 2mL lysing kit (2 mL reinforced empty vials with 2.8 mm stainless steel beads from Bertin Technologies, Montigny-le-Bretonneux, France, ref : P000917-LYSK0-A.0)

 

Decontamination and Pulverization

To remove undesired contaminants from the hair surface, 1 mL of methanol was added to the samples, and it was soaked for 5 minutes at room temperature. The washing solution was discarded then samples were dried at 60°C / convection for 30 minutes. Samples were pulverized into a fine powder (3X 60sec at 6500 rpm, 15 seconds pause time) using Precellys 24 Touch system (Figure 3).

Sample Extraction

Samples were extracted as follows:

Figure 3 – Hair pulverization system (Precellys 24 Touch) from Bertin Technologies, Montigny-le-Bretonneux, France.

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:

MS/MS

MS model: Q-Trap 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 268.2 → 183.2 10
Nordiazepam 271.1 → 140.1 27
7-Aminoflunitrazepam 284.0 → 226.0 50
Diazepam 285.1 → 154.1 32
7-Amino Clonazepam 286.1 → 222.2 30
Oxazepam 287.1 → 241.1 30
7-Amino Clonazepam-d4 290.1 → 226.0 30
7-Amino Flunitrazepam-d7 291.0 → 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 → 264.1 30
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) Hydroxyalprazolam 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 342.2 → 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 (pg/mg hair)

A screening cut-off of 500 pg/mg hair is reached for 7-aminoflunitrazepam, 7-aminoclonazepam and nordiazepam. For all other drugs, a screening cut-off of 250 pg/mg hair is obtained.

 

Precision

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 2 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 2 – Inter-Run Precision for 2-Hydroxyethylflurazepam
QC-L CO QC-H
Conc (pg/mg hair) 125 250 500
N 15 15 15
Mean (pg/mg hair) 123.7 246.2 505.6
SD 17.5 15.2 21.3
%CV 14.1 6.2 4.2
Mean – 2SD (pg/mg hair) 88.8 215.7 463.1
Mean + 2SD (pg/mg hair) 158.7 276.7 548.2

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.

Figure 4 – Intra-Run Precision Curves for 2-Hydroxyethylflurazepam

Multi-Matrix Validation

Hair samples are collected from ten different volunteers. Samples are screened to verify the presence of each analyte (all samples were negative). Drugs are spiked 200% cut-off (QC-H) and screened as unknown to verify the method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.

 

Spiked Sample

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 3 shows the analysis result of non-spiked/spiked samples for 2-Hydroxyethylflurazepam.

Table 3 – 2-Hydroxyethylflurazepam results
 
 
LC-MS/MS
Yes No
LDTD-MS/MS Yes TP=10 FP=0
No FN=0 TN=10

 

Validation results are reported in Table 4 for 2-Hydroxyethylflurazepam. Similar results are obtained for the other drugs.

Table 6 – Validation results for 2-hydroxyethylflurazepam
Parameters 2-Hydroxyethylflurazepam
Sensitivity (%) 100
Specificity (%) 100
PPV (%) 100
NPV (%) 100
Accuracy (%) 100

Conclusion

The Precellys®24 Touch can be successfully used to pulverize hair samples for mass spectrometry drug analysis. The Precellys 24 Touch combined with the Luxon Ion Source® and Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) benzodiazepine panel screening in hair using a simple sample preparation method.