Screening of SAMHSA Drug Panel in Urine

Authors: Mégane Moreau, Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, SAMHSA, Urine, LDTD-MS/MS
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Introduction

The SAMHSA Drug Panel was established to evaluate the presence of the most common drugs of abuse and their metabolites in biological matrices. The panel contains stimulants such as cocaine and amphetamines, depressants such as opiates and opioids, and perturbators such as PCP. Urine, a biological matrix strong in information concerning the health of a patient or the consumption of drugs, is the matrix used in this application note.

Our goal with this application note is to develop a rapid screening method for the SAMHSA panel in human urine using the LDTD technology.

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. Twelve 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.

Sample Preparation Method

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:

MS/MS Positive analysis Negative analysis
MS model: QTrap® System 5500, Sciex QTrap® System 5500, Sciex
Ion Spray Voltage 6000 -6000
Temp., GS1 and GS2 0 0
CUR 20 20
Scan Time 3 – 20 msec 3 – 20 msec
Analysis Method Positive MRM mode Negative MRM mode

 

Table 1 – Positive MRM transitions for LDTD-MS/MS
Drugs Transition CE
6-monoacetylmorphine 328.1 → 165.0 45
6-monoacetylmorphine-d6 334.0 → 165.0 45
Amphetamine 136.0 → 119.0 15
Amphetamine-d5 141.1 → 124.0 15
Benzoylecgonine 290.1 → 168.0 20
Benzoylecgonine-d8 298.0 → 171.1 30
MDA 180.1 → 133.1 10
MDA-d5 185.1 → 138.3 10
MDMA 194.0 → 163.0 20
MDMA-d5 199.2 → 165.1 20
Methamphetamine 150.1 → 119.0 15
Methamphetamine-d5 159.1 → 125.0 15
Morphine 286.1 → 152.0 65
Morphine-d9 292.1 → 152.0 65
Codeine 300.1 → 152.0 70
Codeine-d6 306.2 → 152.0 70
Oxycodone 316.1 → 241.1 32
Oxycodone-d6 322.1 → 247.0 35
Oxymorphone 302.1 → 227.1 30
Phencyclidine 244.2 → 159.2 15
Phencyclidine-d5 249.0 → 164.1 15

 

Table 2 – Negative MRM transitions for LDTD-MS/MS
Drugs Transition CE
Delta-9-THCC 343.2 → 245.1 -35
Delta-9-THCC-d9 352.3→ 254.1 -35

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 3.

Table 3 – Analytes and cut-offs
Analyte Cut-off (ng/mL)
Marijuana (delta-9-THCC) 50
Cocaine (Benzoylecgonine) 150
Codeine/Morphine 300
Hydrocodone/Hydromorphone 300
Oxycodone/Oxymorphone 100
Heroin (6-Acetylmorphine) 10
PCP (Phencyclidine) 25
Amphetamine/Methamphetamine 500
MDA/MDMA 500

 

Precision / Accuracy

Three-point screening curve and two QCs (QC-0.5X and QC-2X) were prepared in synthetic urine and used to validate the method. The peak area against the internal standard ratio was used to normalize the signal. Replicate extractions are deposited on a LazWellTM plate and dried before analysis.
The following acceptance criteria were used:

For the inter-run precision/accuracy experiment, each fortified sample set is analysed in triplicate on five different days. Table 4 shows the inter-run precision and accuracy results. For MDMA, the %CV and %Bias was below 20 %. All QC-0.5X were detected as negative and all QC-2X were detected as positive. Similar results were obtained for the other drugs on the panel.

 

Table 4 – Inter-Run Precision and Accuracy for MDMA
MDMA 1 X 2 X 3 X
Conc (ng/mL) 500 1000 2500
N 25 25 25
Mean (ng/mL) 520.6 945.0 2534.3
%CV 4.3 4.5 2.6
%Bias 4.1 -5.5 1.4

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 1 day, sample extracts are spotted on a LazWell™ plate, dried and analyzed by LDTD-MS/MS. The precision and accuracy results of QCs are reported in Figure 3. 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 2 hours before analysis. The precision and accuracy results of QCs are reported in Figure 3. All the results are within the acceptable criteria range for 2 hours at room temperature.

A)Precision of Intra-Run Assay of 2-Hydroxyethulflurazepam

B)

Precision of Intra-Run Assay of 2-Hydroxyethulflurazepam
Figure 3 – Wet and Dry Stability A) Precision of the calibrator 1X; B) Accuracy of the calibrator 1X

 

Multi-Matrix Validation

Urines are collected from twenty different volunteers. Samples are screened to cross-verify with LC-MS/MS to assess the method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.

 

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 5 shows the analysis results of 23 spiked matrices for THCC.

Table 5 – THCC results
 
 
LC-MS/MS
Yes No
LDTD-MS/MS Yes TP=2 FP=0
No FN=0 TN=21

 

Validation results are reported in Figure 6 for THCC. Similar results are obtained for the other drugs. No false negative was observed.

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

Conclusion

The Luxon Ion Source® combined with the Sciex QTrap® 5500 mass spectrometer system enables high-throughput analysis of the SAMHSA drug panel in urine.