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.
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).
Sample Extraction
To analyze drugs of abuse in nail clippings, the samples were extracted as follows1:
- Add 1 mL of the internal standard solution (acetonitrile:water / 75:25) in a 2 mL vial.
- For the screening curve: 25 µL of the working solution of the standard were added.
- Vials were capped, mixed and incubated at room temperature for 1.5 hours (horizontal mixing at 500 rpm).
- Add 0.5 mL of extraction buffer in extraction vial.
- Vortex.
- Centrifuge for 2 minutes at 5000 rpm.
- Mix 10 µL of desorption buffer with 90 µL of the upper layer phase.
- Spot 6 µL of the mixture onto a LazWell™96 plate
- Dry 4 minutes at 40°C
LDTD®-MS/MS Parameters
LDTD
Model: Luxon S-960, Phytronix
Carrier gas: 6.0 L/min (air)
Laser pattern:
- 6-second ramp to 65% power
- Hold 1 second at 65% power
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
| 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:
- Each standard concentration must not exceed 20% CV
- Each standard concentration must be ±20% of the nominal value (%Biais).
- QC-0.5X cut-off must be detected as negative.
- QC-2X cut-off must be detected as positive.
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.
| 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.
| 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.
| |
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.
| 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.