Screening of SAMHSA Drug Panel in Oral Fluids

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

In 2019, the US Department of Health and Human Services (via the SAMHSA agency) has established scientific and technical guidelines for federal workplace drug testing programs in oral fluids (Federal Register / Vol. 84, No. 207, 2019). Our goal for this application note is to use an automated sample preparation method for the SAMHSA drug panel in oral fluids using a single operation in LDTD-MS/MS.

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 quick and simple sample preparation. Fourteen drugs are 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

Oral fluids were collected using the Quantisal® device. After the collection of the oral fluid, the pad is transferred into a tube containing an extraction buffer. During this process, oral fluids are diluted by a factor of 4.

 

Figure 3 – Automated extraction system – Azeo Liquid Handler

Automated Sample Extraction (SALLE)

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

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon SH-960, Phytronix

Carrier gas: 6.0 L/min (air) with water infusion at 20 µL/sec

Laser pattern:

MS/MS

MS model: LCMS-8060, Shimadzu

Scan Time: 5 msec

Ionization: APCI

Analysis Method: Positive MRM mode

 

Table 1 – MRM transitions for LDTD-MS/MS
Drugs Transition CE
Amphetamine 136.1 → 119.1 12
Amphetamine-d5 141.1 → 124.1 12
Methamphetamine 150.1 → 119.1 15
Methamphetamine-d9 159.1 → 125.1 15
MDA 180.1 → 133.0 20
MDA-d5 185.1 → 138.1 20
MDMA 194.1 → 133.1 25
MDMA-d5 199.2 → 136.1 25
PCP 244.2 → 159.1 20
PCP-d5 249.3 → 164.0 20
MOR / HYM 286.1 → 152.0 70
MOR-d6 292.1 → 152.0 70
COD / HYC 300.1 → 152.0 70
COD-d6 306.1 → 152.0 70
Cocaine 304.1 → 182.1 25
Cocaine-d3 307.2 → 185.2 25
OXM 302.1 → 227.0 40
OXC 316.1 → 241.0 35
OXC-d6 322.2 → 247.0 35
6-AM 328.1 → 165.0 50
6-AM-d6 334.1 → 165.0 50
THC 315.2 → 193.1 25
THC-d3 318.2 → 196.1 25

 

Results and Discussion

Initial Cut-Off Test (ng/mL)

A drug list and screening cut-offs suggested in the SAMHSA guidelines can be found in Table 2.

Table 2 – Analytes cut-offs
Analyte Cut-off (ng/mL)
Marijuana (THC) 4
Cocaine 15
Codeine / Morphine 30
Hydrocodone / Hydromorphone 30
Oxycodone / Oxymorphone 30
6-Acetylmorphine 4
Phencyclidine 10
Amphetamine / Methamphetamine 50
MDA / MDMA 50

 

Data Preparation Process

Data acquisition systems of mass spectrometers were not designed to deal with signals of only a few seconds per sample. The synchronization sequence adds 6 to more than 15 seconds between each sample. To bypass this, all samples are acquired in a single file (Figure 4). To allow the analysis of such data, Cascade software is designed to detect, split, and integrate every sample peak acquired in a single file.

Figure 4 – Single file mass spectrometer data for 96 samples. Cocaine-d3 transition.

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 3 shows the inter-run precision results. No overlapping at the cut-off is observed for THC and the %CV was below 20%. Similar results are obtained for the other drugs in the panel.

 

Table 3 – Inter-Run Precision for THC
QC-L CO QC-H
Conc (ng/mL) 2 4 8
N 15 15 15
Mean (ng/mL) 2.01 3.93 8.02
SD 0.271 0.545 0.909
%CV 13.5 13.7 11.3
Mean – 2SD (ng/mL) 1.47 2.88 6.2
Mean + 2SD (ng/mL) 2.55 5.06 9.84

Wet Stability of Sample Extracts

Following the extraction, the upper layer was mixed with a desorption solution and kept at 4°C in closed containers. After 1 day, sample extracts are spotted on a LazWell™ plate, dried and analyzed. Precision of QC samples are reported in Table 4. All the results are within the acceptable criteria range for 1 day at 4°C.

Dry Stability of Samples Spotted on LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 1 hour before analysis. The precision results of QC samples are reported in Table 4. All the results are within the acceptable criteria range for 1 hour at room temperature.

Table 4 – Wet and Dry Stability of THC
Parameters Dry Stability
(1 hour / RT)
Wet Stability
(1 day / 4°C)
QC QC-L QC-M QC-H QC-L QC-M QC-H
Conc (ng/mL) 2 4 8 2 4 8
N 3 3 3 3 3 3
Mean (ng/mL) 1.93 4.21 7.86 2.10 3.70 8.20
%CV 9.9 5.2 11.0 14.2 3.0 5.8

Multi-Matrix Validation

Oral fluids are collected from eight different volunteers. Samples are screened to verify the presence of each analyte (all samples were negative). Drugs are spiked at 50% cut-off (QC-L) and 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 5 shows the analysis results of 16 spiked matrices for THC.

Table 5 – THC results
 
 
LC-MS/MS
Yes No
LDTD-MS/MS Yes TP=8 FP=0
No FN=0 TN=8

 

Validation results are reported in Table 6 for THC. Similar results are obtained for the other drugs.

Table 6 – Validation results for THC
Parameters THC
Sensitivity (%) 100
Specificity (%) 100
PPV (%) 100
NPV (%) 100
Accuracy (%) 100

Conclusion

Luxon Ion Source® combined to a Shimadzu LCMS-8060 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of the SAMHSA drug panel in oral fluids using a simple and automated sample preparation method.