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SPE Method to Eliminate Matrix Effect in the LC/MS (TOF) Analysis of Illicit NBOMe Synthetic Hallucinogens from Urine

By: Craig Aurand, David S. Bell, Reporter US, Volume 33.3

Supel™-Select HLB plates provided excellent analyte recovery of these difficult compounds from urine samples with subsequent LC/MS (TOF) analysis on Ascentis® Express Fused-Core® 2.0 µm C18 columns. The extracts were free of endogenous matrix that can interfere with quantitation, decrease sample throughput and reduce column lifetime.

N-methoxybenzyl derivative drugs (25l-NBOMe) and other similar substituted phenethylamines behave as psychedelic hallucinogens. In order to evade drug laws and provide novel experiences, illicit drug manufacturers are constantly developing new compounds. With the proliferation of these synthetic designer drugs, LC/MS analysis has become an increasingly important tool for forensic laboratories. The high specificity of LC/MS is relied upon for detecting illicit compounds. However, there are still issues related to method robustness and matrix interference that must be addressed. This has driven the need for developing stronger analytical methods that can accommodate an ever changing list of synthetic compounds, along with heightened awareness of the need for efficient sample preparation.

The goal of this study was to develop a sensitive LC/MS method for the analysis of NBOMe synthetic hallucinogens from urine samples. The study focused on the development of a simple LC/MS (TOF) method, including solid phase extraction (SPE) sample preparation using Supel-Select HLB plates, for the direct analysis of eight different analogs of NBOMe hallucinogens.

Chromatographic (LC/MS - TOF) Conditions
The chemical structures of the eight NBOMe hallucinogens used in this study are shown in Figure 1. Initial evaluation was conducted using a mixture of NBOMe compounds to establish chromatographic conditions on an Ascentis Express C18 column. The chromatographic conditions along with a representative chromatogram are shown in Figure 2.

Structures of NBOMe Compounds

Figure 1. Structures of NBOMe Compounds

LC/MS (TOF) Analysis of NBOMe Hallucinogens on Ascentis Express C18

Figure 2. LC/MS (TOF) Analysis of NBOMe Hallucinogens on Ascentis Express C18

Conditions
column:
Ascentis Express C18, 10 cm × 3.0 mm I.D., 2.0 µm particles (Product No. 50819-U); mobile phase: (A) 20 mM ammonium acetate, pH 4.0 with acetic acid; (B) methanol; 60:40 (A:B); flow rate: 0.8 mL/min; column temp: 50 °C; pressure: 7600 psi; detector: ESI(+), full scan; injection: 1 µL; sample: each compound 300 ng/mL in water:methanol (50:50); system: Agilent® 1290 Infinity with 6210 TOF

 

Method Calibration
Calibration curves were developed using a standard composed of eight individual NBOMe reference standard materials supplied by Cerilliant. Levels of 10, 20, 50, 100, 200, and 300 ng/mL were prepared in methanol:water (50:50). Analyte detection was conducted using accurate mass measurement. Figure 3 depicts the linear calibration curve for 25I-NBOH. Table 1 depicts the correlation coefficients for all eight standard compounds.

LC/MS (TOF) Standard Calibration Curve of NBOMe compound 25I-NBOH on Ascentis Express 2 µm C18

Figure 3. LC/MS (TOF) Standard Calibration Curve of NBOMe compound 25I-NBOH on Ascentis Express 2 µm C18

Table 1. Linear Calibration Coefficients of Analytes Across the 10-300 ng/mL Range

Compound Correlation Coefficient (r2)
25T-NBOMe 0.99421
25C-NBOMe 0.9941
25I-NBOH 0.99863
25D-NBOMe 0.98982
25B-NBOMe 0.98999
25I-NBF 0.9927
25I-NBMD 0.98987
25I-NBOMe 0.97963

 

Supel-Select HLB Operating Principles
Supel-Select HLB SPE particles are hydrophilic-modified styrene polymer beads. The base polymer is highly effective at extracting hydrophobic compounds from aqueous samples with analyte retention predominately based on reversed-phase interactions. The hydrophilic component of the polymer makes it amenable for the extraction of polar compounds. It is therefore suitable for extracting an extensive range of analyte polarities from aqueous matrices.

Recovery of Analytes from Standard Solution and Spiked Urine
Method development with the Supel-Select HLB 96-well plate began with establishing recovery of the eight NBOMe compounds from standard solutions in water. A 1.0 mL aliquot of standard solution (100 ng/mL each component) was processed through the Supel-Select HLB-96-well plate following the conditions outlined in Table 2. Vacuum was used to process the samples through the Supel-Select HLB 96-well plate. Recovery was greater than 70% for all compounds tested. Spiked human urine samples processed using the Supel-Select HLB SPE plates were free of matrix interference and exhibited excellent recovery, in good agreement with the standard solutions (Table 3).

 

Table 2. SPE Conditions

SPE Plate: Supel-Select HLB SPE, 96-wellplate, 30 mg/well (Product No. 575661-U)
Conditioning: 1 mL methanol followed by 1 mL water
Load: 1 mL urine sample
Wash: 1 mL water followed by 1 mL water:methanol (75:25)
Elute: 1.5 mL methanol
Evaporate sample and reconstitute in 1 mL of water:methanol (50:50)

 

 

Table 3. Recovery of NBOMe Compounds as Standards and from Spiked Urine

  25I-NBOH 25T-NBOMe 25C-NBOMe 25D-NBOMe 25I-NBF
Standard Solution % 67.5 72.0 78.7 79.6 79.6
CV 6.7 6.3 5.1 4.1 4.0
Spiked Urine % 84.6 80.3 86.0 84.1 88.6
CV 4.1 4.2 4.0 2.8 2.7
 
  25B-NBOMe 25I-NBMD 25I-NBOMe    
Standard Solution % 78.7 75.7 80.9    
CV 5.3 4.4 5.8    
Spiked Urine % 88.2 90.2 96.6    
CV 2.2 2.2 2.1    

Conclusion

The Supel-Select HLB 96-well plate was shown to be an effective method to remove matrix interference in urine samples prior to analysis by LC/MS (TOF). Recovery of all eight NBOMe compounds from human urine samples was greater than 80%. The processed samples were free of matrix interference. The generic methodology of the Supel- Select HLB allows sufficient sample cleanup while also enabling selective extraction of a broad range of target analytes. The Ascentis Express 2.0 µm C18 column produced efficient separation of all eight NBOMe compounds resulting in a sensitive LC/MS method.

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