Rapid Screening of Xylazine in Urine

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

 

Salt-Assisted Liquid-Liquid Extraction

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

Total run time: 9 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Q1 Q3 Time
(msec)
DP
(V)
CE (V)
Xylazine 221.1 90 50 80 20
Fentanyl-d5 342.2 188.1 50 80 20

 

Results and Discussion

 

Screening range (µg/mL)

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

Table 2 – Xylazine calibrators concentration

Analyte Cal 1
(ng/mL)
Cal 2
(ng/mL)
Cal 3
(ng/mL)
Xylazine 25 50 125

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:

Table 2 – Xylazine calibrators concentration

 

Run 1 0.99533
Run 2 0.99533
Run 3 0.99407
Run 4 0.99213

 Precision and Accuracy

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

Table 4 – Inter-Run Precision and Accuracy

Cal 1 Cal 2 Cal 3
Nominal (ng/mL) 25 50 125
N 12 12 12
Mean (ng/mL) 24.8 51.9 123.9
%CV 12.2 8.7 7.9
%Bias -0.6 3.8 -0.9

 

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.

Results are presented in Table 5.

Table 5 – Intra-Run results for Xylazine samples

Run 1 Run 2 Run 3 Run 4
Blank Negative Negative Negative Negative
Conc (ng/mL) 25 25 25 25
N 3 3 3 3
%CV 8.1 12.8 9.0 7.8
QC-0.5X Negative  Negative  Negative  Negative
QC-2X Positive Positive Positive Positive

 

Blank Interference Evaluation

10 blank matrices were analyzed to evaluate the blank interference.

 

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

Dry stability
(1 day / RT)
Wet stability
(14 days / 4°C)
Blank Negative Negative
Conc. (ng/mL) 25 25
N 3 3
%CV 2.4 3.1
QC-0.5X Negative Negative
QC-2X Positive Positive

 

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.