Drug Screening in Whole Blood for Driving Under Influence Cases

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

Impaired driving is a public health and safety concern. Toxicological testing is a critical part of these investigations. Academy standards board (ASB) published a guideline of the minimum requirements for target analytes and analytical sensitivity (ANSI/ASB standard 120, 1st Ed. 2021). Samples from these investigations are sent to crime labs, however, the analysis time per sample and the volume of requests creates delays. For forensic analysis laboratories, an improvement in the speed of sample analysis must be made. Indeed, in 2014, more than 500,000 samples were backlogged1.

Our goal for this application note is to use a simple sample preparation method and a fast analysis technique for drug screening in whole blood. Based on their chemical properties, two panels were used with a specific sample preparation.

The LDTD-MS/MS system offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a simple sample preparation. Panel 1 was extracted using a Salt Assisted Liquid-Liquid Extraction (SALLE) approach and a Liquid-Liquid Extraction (LLE) was used for Panel 2. The drugs present in the first panel have a greater affinity for acetonitrile while the drugs present in the second panel have a greater affinity for MTBE. This is why the drugs under study have been divided into two panels according to their affinity with the extraction solvent.

Each panel was analyzed simultaneously with quantitative screening results obtained in less than 8 seconds per sample and specific cut-off values were attained for each drug.

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

Whole blood 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

Ionization: APCI

Analysis Method: Positive MRM mode

 

Table 1 – MRM transitions for SALLE extraction with LDTD-MS/MS
Drugs Transition CE
Methamphetamine 150.1 → 119.1 15
Methamphetamine-d5 155.1 → 121.1 15
MDA 180.1 → 133.1 20
MDA-d5 185.1 → 138.1 20
Tramadol 264.2 → 58.2 50
Oxazepam 292.1 → 246.1 30
Benzoylecgonine 290.1 → 168.2 30
Oxazepam-d5 292.1 → 246.1 30
Benzoylecgonine-d3 293.1 → 171.1 30
Temazepam 301.1 → 255.1 25
Temazepam-d5 306.1 → 260.1 25
Methadone 310.2 → 265.2 20
Methadone-d9 319.3 → 268.2 20
Lorazepam 321.0 → 275.0 30
Buprenorphine (-H2O) 450.3 → 418.3 25
Buprenorphine-d4 (-H2O) 454.3 → 422.3 25

 

Sample Extraction (LLE): Panel 2

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

Ionization: APCI

Analysis Method: MRM mode

 

Table 2 – Positive MRM transitions for LLE extraction with LDTD-MS/MS
Drugs Transition CE
Amphetamine 136.1 → 119.1 12
Amphetamine-d5 141.1 → 96.0 12
MDMA 194.1 → 133.0 25
MDMA-d5 199.2 → 165.1 20
Carisoprodol 261.2 → 176.2 25
Carisoprodol-d7 268.2 → 183.2 25
Nordiazepam 271.1 → 140.1 27
Diazepam 285.1 → 154.1 32
Morphine/HYM/Norhydrocodone 286.1 → 152.0 75
Oxazepam-d5 292.1 → 246.1 40
Morphine-d6 292.1 → 152.0 75
Codeine/Hydrocodone 300.2 → 152.0 75
Temazepam-d5 306.1 → 260.1 40
Codeine-d6 306.2 → 260.1 40
Zolpidem 308.1 → 236.1 35
Alprazolam 309.1 → 274.1 35
Alprazolam-d5 314.1→ 286.1 45
Clonazepam 316.0 → 214.0 50
Oxycodone 316.2 → 241.0 35
Oxycodone-d6 322.2 → 247.0 25
Fentanyl 337.2 → 188.1 25
Fentanyl-d5 342.2 → 188.1 35

 

Table 3 – Negative MRM transitions for LLE extraction with LDTD-MS/MS
Drugs Transition CE
THCC 343.3 → 245.2 -45
THCC-d9 352.3 → 254.2 -45

 

Results and Discussion

Initial Cut-Off Test (ng/mL)

A drug list and screening cut-offs requested by ANSI/ASB standard 120 guidelines can be found in Table 4.

Table 4 – Analytes and cut-offs
Analyte Cut-off (ng/mL) Analyte Cut-off (ng/mL)
Carboxy-THC 10 Benzoylecgonine 50
Alprazolam 10 Fentanyl 1
Clonazepam 15 Codeine 10
Lorazepam 15 Hydrocodone 10
Diazepam 50 Morphine 10
Nordiazepam 50 Oxycodone 10
Oxazepam 50 Amphetamine 20
Temazepam 50 Methamphetamine 20
MDA 25 MDMA 25
Carisoprodol 1000 Zolpidem 10
Methadone 50 Buprenorphine 1
Tramadol 100

 

Precision / Accuracy

Three-point screening curves and two QCs (QC-0.5X and QC-2X) were prepared in negative nail powder and used to validate the method. The peak area against the internal standard (IS) ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell™ plate and dried before analysis.

The following acceptance criteria were used:

For the inter-run precision/accuracy experiment, each fortified sample set is analyzed in triplicate on five different days. Table 2 shows the inter-run precision and accuracy results. For temazepam, the %CV and %Bias was below 20%. All QC-0.5X were detected as negative and QC-2X detected as positive. Similar results are obtained for the other drugs in the panel.

 

Table 5 – Inter-Run Precision / Accuracy
Temazepam S1 S2 S3
Conc (ng/mL) 50 100 250
N 15 15 15
Mean (ng/mL) 50.3 99.2 250.5
%CV 8.0 7.8 7.2
%Bias 0.6 -0.8 0.2

Wet Stability of Sample Extracts

Following the extraction, sample extracts were kept at room temperature in closed containers. After 1 day, sample extracts were spotted on a LazWell™ plate, dried and analyzed. Precision and accuracy of standard are reported in Figure 4 and 5. All the results are within the acceptable criteria range for 1 day at room temperature.

Dry Stability of Samples Spotted on LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 75 minutes before analysis. The precision and accuracy results of standard samples are reported in Figure 4 and 5. All the results are within the acceptable criteria range for 75 minutes at room temperature.

 

Figure 4 – Precision of sample after stability tests
Figure 5 – Accuracy of sample after stability tests

 

Blank Matrix Interference

Seven different blank matrices were extracted and analyzed to evaluate the drug signal. The following table (Table 6) presents the results of the blank matrix interference. Similar results are obtained for the other drugs.

 

Table 6 – Evaluation of blank matrix interference for Buprenorphine, Lorazepam and MDA
Buprenorphine Lorazepam MDA
Matrix 1 Negative Negative Negative
Matrix 2 Negative Negative Negative
Matrix 3 Negative Negative Negative
Matrix 4 Negative Negative Negative
Matrix 5 Negative Negative Negative
Matrix 6 Negative Negative Negative
Matrix 7 Negative Negative Negative

Multi-Matrix Validation

Negative whole blood (EDTA-K2) was collected from 6 volunteers. Samples are analyzed unspiked, spiked at QC-0.5x and QC-2X level and screened using LDTD-MS/MS method. The method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy are verified as followed:

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 7 shows the analysis results of 18 spiked samples for Temazepam.

Table 7 – Temazepam results
 
 
LC-MS/MS
Yes No
LDTD-MS/MS Yes TP=6 FP=0
No FN=0 TN=12

 

Validation results are reported in Table 8 for temazepam. Similar results are obtained for the other drugs.

Table 8 – Validation results for Temazepam
Parameters Temazepam
Sensitivity 1
Specificity 1
PPV 1
NPV 1
Accuracy 1

Conclusion

Luxon Ion Source® combined to a Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) drug screening in whole blood using a simple and efficient sample preparation method.