44 February / March 2017
sufentanil-d5, butyryl fentanyl, acetyl fentanyl, acetyl fentanyl- 13C were purchased from Cerilliant® (Round Rock, TX). Blank human saliva was purchased from BioReclamation (NY).
BioSPME extraction procedure. SPME LC Tips were conditioned within 1 mL of 50:50 methanol for 30 min with 800 rpm agitation, followed by rinse in 1 mL of water for 10 seconds with agitation. The fibres were then placed in 1 mL of spiked saliva samples and agitated for 30 min at 800 rpm using an orbital shaker. The fibres were then rinsed with 1 mL of water for ~ 10 seconds with agitation and analysed directly on the DART-MS.
Blank human saliva was fortified with the analytes of interest over a concentration range of 50 to 2500 ng/mL in order to prepare a matrix matched calibration curve. In addition, six separate fibres were extracted in individual blank human saliva samples fortified at concentrations of 100 ng/mL, 500 ng/mL, and 1000 ng/mL. A 30 minute equilibration time was allowed after matrix spiking. For matrix effect evaluations, fibres were also extracted in blank saliva samples, blank water samples, and water samples fortified at 100 ng/mL (n=6).
Analyte Fentanyl
Fentanyl-d5 Alfentanil
Sufentanil Sufentanil-d5
Butyryl fentanyl Acetyl fentanyl Acetyl fentanyl-13
EIC (m/z) 337.4 342.4
417.4^ 387.3 392.4
C
351.4^ 323.3 329.3
Table 2. Compound specific MS parameters. Results and Discussion
After the extraction period, the fibres were passed in front of the DART source. The results for twelve of the fibres were stored in a single data file. Full scan data was collected and then extracted ion chromatograms were generated for each analyte and internal standard. An example data file with 12 fibre responses for butyryl fentanyl is shown in Figure 3.
Figure 3. Extracted ion response for butyryl fentanyl in saliva
Figure 2. Schematic of DART-MS Set-up with BioSPME fibre tips.
DART®-MS analysis. Samples were analysed using an IonSense® DART®-SVP coupled to a Waters QDa® mass spectrometer. Figure 2 depicts the DART source interfaced to the QDa with the BioSPME fibres positioned for analysis. The MS source and compound dependent parameters are displayed in Tables 1 and 2.
Parameter
DART Temp. (°C) Polarity
Cone (V) Gas
Rail Speed (mm/sec) Frequency (Hz) Scan Type (SIM)
Setting 300
Positive 15
Helium 0.3 20
100-500 amu Table 1. DART-MS source settings. Figure 7. Calibration curve for butyryl fentanyl in saliva
The method demonstrated reproducible extraction efficiencies with accuracies ranging from 81-120% for all analytes and relative standard deviations (% RSD) ranging from 1.9-7.0%. Table 3 shows the average accuracies and % RSD’s
Calibration curves constructed from the extracted blank saliva samples that were fortified at concentrations ranging 50- 2500 ng/mL were used to determine the average accuracy for measurement of each analyte for the 100 ng/mL spiked samples. Using linear regression, calibration curves demonstrated linearity from 50 – 2500 ng/mL in saliva with coeffecients of determination (R2) values greater than 0.985. An example calibration curve for butyryl fentanyl is given in Figure 4.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
