Rapid Screening of Xylazine in Urine

Introduction

Xylazine, also know as tranq or zombie drug, is a new muscular relaxant widely used to adulterate drugs on the street. This tranquilizer is linked to overdose deaths. In Canada and the United States, xylazine is authorized only in veterinary medicine. The presence of this drug on the black market presents a public health threat. In fact, this muscle relaxant is known to cause respiratory distress and cardiac slowing. In addition to causing potentially fatal effects, naloxone, used in cases of overdose, is powerless against xylazine. A rapid analysis of consumers’ urine would make it possible to quickly establish the presence of xylazine in certain areas to implement means of control (analysis by infrared spectroscopy in supervised consumption centers) and prevention.

Our goal for this application note is to use a simple sample preparation method for the screening of xylazine in urine using a single operation in LDTD-MS/MS.

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. Xylazine screening results were obtained in less than 12 seconds per sample.

Sample Preparation Method

Hydrolysis

• 5 µL of Internal standard is added into a Micro Centrifuge Tube of 1.5 mL using an automatic pipet.
• 75 µL of Urine sample were transferred to the 1.5 mL Micro Centrifuge Tube (containing IS) using an automatic pipet.
  • Mix at 1000 rpm for 30 seconds.
• 5 µL of β-Glucuronidase-RT Enzyme/Hydrolysis buffer (1:2) is added to each sample using an automatic pipet.
  • Mix at 1000 rpm for 30 seconds and incubate at room temperature for 15 minutes.

Salt-Assisted Liquid-Liquid Extraction

• 150 µL Extraction buffer (1 M K₂HPO₄: NaCl (sat) (1:1)) is added into to the 1.5 mL Micro Centrifuge Tube using an automatic pipette.
• 300 µL Acetonitrile is added into the Micro Centrifuge Tube 1.5 mL using an automatic pipette.
  • Cap and mix at 1000 rpm for 30 seconds.
  • Centrifuge at 13500 rpm for 3 minutes for phase separation.
• Transfer 15 µL of upper layer phase into a new Microcentrifuge tube using automatic pipette and add 45 µL of desorption buffer (5 mM KH₂PO₄, 25 mg/mL BSA in water).
  • Vortex
• Spot 6 µL of mixture onto a LazWell™96 plate using an automatic pipette.
  • Dry 8 minutes at 40°C using the Aura LazWell Dryer

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:

• 6-second ramp to 85% power
• Hold 1-second at 85% power

MS/MS

MS model: QTrap® System 5500, Sciex

Total run time: 9 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Results and Discussion

Screening range (µg/mL)

The screening range for xylazine can be found in Table 2.

Table 2 – Xylazine calibrators concentration

Validation Test

For the screening of xylazine in urine, a complete validation was carried out. Linearity, precision, accuracy, stability, and blank interference were evaluated.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

• Linear regression (r) must be ≥ 0.99

Table 2 – Xylazine calibrators concentration

 Precision and Accuracy

Inter-run validation tests were carried out using a tree points calibration curve. Each standard was analysed in triplicate.

• For the precision acceptance criterion, %CV shall not exceed 20% for at least 66.7% of each standard.
• For the accuracy acceptance criterion, %Bias shall not exceed 20 % for at least 66.7% of each standard.

Table 4 – Inter-Run Precision and Accuracy

Run acceptance criteria for intra-run

Intra-run validation tests were carried out using a one-point calibration curve and linear through zero regression. Each standard was analyzed in triplicate.

• Blank samples must be detected as negative.
• At Cal-1X concentration, %CV must not exceed 20% for at least 66.7% of this standard.
• At QC-0.5X, at least 66.7% of sample must be detected as negative.
• At QC-2X, 66.7% of sample must be detected as positive.

Results are presented in Table 5.

Table 5 – Intra-Run results for Xylazine samples

Blank Interference Evaluation

10 blank matrices were analyzed to evaluate the blank interference.

• At xylazine signal, all matrices must be, and were detected as negative.
• At fentanyl-d₅ (Internal standard) signal, all matrices must have an interference peak lower than 20% (peak area) of the Cal-1. All matrices blank interference at IS signal were lower than 2.6%.

Dry Stability of Samples Spotted in LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 1 day before analysis. Acceptance criteria are the same as the run acceptance criteria. Results are presented in Table 6. All the results are within the acceptable criteria range for 1 day at room temperature.

Wet Stability of Samples Spotted in LazWell™

Following the extraction, sample extracts are kept at 4°C in closed containers. After 14 days, sample extracts are spotted on a LazWell™ plate, dried and analyzed. Acceptance criteria are the same as the run acceptance criteria. Results are presented in Table 6. All the results are within the acceptable criteria range for 14 days at 4°C.

Table 6 – Wet and Dry Stability of Xylazine samples

Conclusion

The Luxon Ion Source® combined with the Sciex QTrap^® 5500 mass spectrometer system enables the rapid analysis of xylazine in urine. This analysis method can thus be used in toxicological applications to assess intoxication and the presence of xylazine in the territory.