15 Drugs of Abuse in Nail Clippings

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

The analysis of drugs of abuse in nail clippings is a great alternative to hair analysis, in the case where hair is not available. They provide up to 6 months of drug usage history and are easily collected for testing. Drug of abuse absorption in nails is done by three methods:

 

 

Our goal for this application note is to develop a sample preparation method for a drug panel in nails 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 (Bertin Technologies, Montigny-les-Bretonneux, France.

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. Fifteen drugs (different drug classes) 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

Thirty milligrams of nail clippings 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, p/n : P000917-LYSK0-A.0).

Decontamination and Pulverization

To remove undesired contaminants from the nail surface, 1 mL of dichloromethane (DCM) was added to the samples, and they were soaked for 1 minute at room temperature. The washing solution was then discarded. This washing step was repeated twice. Afterwards, samples were dried at 60°C / convection for 30 minutes. Next, the cleaned nails were pulverized into a fine powder (3 X 60 sec at 6500 rpm, 15 seconds pause time) using a Precellys 24 Touch system (Figure 3).

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

Sample Extraction

To analyze drugs of abuse in nail clippings, the samples were extracted as follows1:

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

Total Run Time: 8 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

 

Table 1 – MRM transitions for LDTD-MS/MS
Transition CE
7-Amino Clonazepam 286.1 → 222.2 30
Alprazolam 309.1 → 274.1 35
Amphetamine 136.1 → 119.1 12
Benzoylecgonine 290.1 → 168.2 30
Clonazepam 316.0 → 214.0 50
Diazepam 285.1 → 154.1 45
Fentanyl 342.2 → 188.1 35
Codein / Hydrocodone 300.2 → 152.0 75
Morphine 286.1 → 152.0 75
Lorazepam 321.0 → 275.0 30
Methamphetamine 150.1 → 119.1 15
Nordiazepam 271.1 → 140.1 27
Oxazepam 287.1 → 104 40
Oxycodone 316.2 → 241.0 35
Temazepam 301.1 → 255.1 25
Oxazepam-d5 292.1 → 109 40
Benzoylecgonine-d3 293.1 → 171.1 30
Amphetamine-d5 141.1 → 124.1 12
Methamphetamine-d5 155.1 → 121.1 15
Morphine-d6 292.1 → 152.0 75
Temazepam-d5 306.1 → 260.1 25
Codeine-d6 306.2 → 152.0 75
Oxycodone-d6 322.2 → 247.0 35
Fentanyl-d5 342.2 → 188.0 35
Alprazolam-d5 314.1 → 286.1 35

 

Results and Discussion

Initial Cut-Off Test (pg/mg nail)

A screening cut-off of 20 pg/mg nail is reached for fentanyl. For all the other drugs, a screening cut-off of 40 pg/mg nail is obtained2.

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 %Biais 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 2 – Inter-Run Precision / Accuracy
Temazepam S1 S2 S3
Conc (pg/mg) 40 80 200
N 15 15 15
Mean (pg/mg) 39.9 80.4 199.8
%CV 4.9 5.8 3.3
%Biais 0.4 0.5 0.1

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. Precision and accuracy of standards are reported in Table 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 60 minutes before analysis. The precision and accuracy results of standard samples are reported Table 3. All the results are within the acceptable criteria range for 60 minutes at room temperature.

Table 3 – Wet and Dry Stability of Temazepam
Parameters Dry Stability
(60 mins / RT)
Wet Stability
(1 day / 4°C)
Conc (pg/mg) 40 80 200 40 80 200
N 3 3 3 3 3 3
Mean (pg/mg) 39.3 81.7 198.9 37.6 86.3 196.1
%CV 4.9 8.4 5.1 5.0 3.4 10.4
%Bias 1.7 2.2 0.5 5.9 7.8 2.0

Multi-Matrix Validation

Negative nail clippings were collected from 6 volunteers. Samples were analyzed without spiking and spiked at QC-0.5x and QC-2X levels and screened using the LDTD-MS/MS method. Results were compared to LC-MS/MS. The method sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy are verified as follows:

 

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 18 spiked samples for temazepam.

Table 4 – 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 5 for temazepam. Similar results are obtained for the other drugs.

Table 5 – Validation results for Temazepam
Parameters Temazepam
Sensitivity 1
Specificity 1
PPV 1
NPV 1
Accuracy 1

Conclusion

The Precellys®24 Touch can be successfully used to pulverize nail 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) screening of different drugs of abuse in nail clippings using a simple sample preparation method.