49
exchange) sorbent, enabling the use of small sample and elution volumes to facilitate LC-MS/MS analysis without the need for evaporation and reconstitution of the extract. Mass spectral detection was performed using a triple stage quadrupole mass spectrometer in SRM mode.
Experimental Materials
Figure 2. (a) Calibration curve from 0 to 100 ng/mL for the LC-MS/MS analysis of THCA in urine following SPE. (b) Chromatogram of the lowest standard ([THCA] = 1.5 ng/mL).
Different approaches have been described for the extraction of THC and its metabolites from urine and oral fl uid, mostly involving Liquid-Liquid Extraction (LLE) [7] or Solid Phase Extraction (SPE) [8-9]. Although these techniques are effi cient at removing matrix components that negatively impact the analysis, they have extensive time and solvent requirements, and the multiple steps can lead to the introduction of error and imprecision.
Micro-elution SPE is based on smaller media bed weights (2 mg) than are typically used for SPE. Although the smaller beds have lower loading capacities than larger beds, methods based on micro-elution SPE benefi t from using less solvent during elution. This reduces the amount of time required for evaporation prior to reconstitution for analysis, or can eliminate this process completely, which has signifi cant advantages to reducing non-specifi c binding which can have adverse effects on the recovery, particularly of larger molecules.
In this article, novel sample preparation approaches are presented for the analysis of THCA in urine as well as the analysis of THC and THCA in oral fl uid that enables reliable detection and quantifi cation of THCA at the low levels required for using this matrix to positively identify cannabis usage. Sample preparation is based on micro-elution solid phase extraction using a polymeric mixed-mode (reversed phase/strong anion
THC, THC-d3, THCA, and THCA-d3 were purchased as solutions in MeOH from Cerilliant Corporation (Round Rock, TX). LC-MS Optima Grade Water and Acetonitrile, and LC/ MS Grade Formic Acid were purchased from Fisher Scientifi c (Waltham, MA) and used as supplied. Human urine and oral fl uid samples were obtained from volunteers and was tested to ensure that it was free of THC and THCA prior to use.
Instrumentation
Separations were performed using a Thermo Scientifi c™ UltiMate™ 3000 RSLC system coupled to either a Thermo Scientifi c™ TSQ Endura™ (urine) or Thermo Scientifi c™ TSQ Quantiva™ (oral fl uid) triple stage quadruple mass spectrometer, each equipped with a heated electro spray ionisation source, HESI II.
Sample Preparation
Solid phase extraction using Thermo Scientifi c™ SOLAµ™ SAX (2 mg/1 mL, 96-well plate) was utilised for both urine and oral fl uid samples. Although different sample pretreatment procedures were utilised for the two matrices, the SPE procedures were the same. The full sample preparation procedure is summarised in Figure 1.
Urine was treated through base hydrolysis with concentrated NaOH prior to solid phase extraction to convert THCA- Glucuronide to its native form
Figure 3. (a) Calibration curve from 0 to 1000 pg/mL for the LC-MS/ MS analysis of THCA in oral fl uid following SPE. (b) Chromatogram of the lowest standard (THCA = 10
pg/mL).
to simplify data analysis and interpretation. Silanised vials were used for the hydrolysis to reduce binding of THCA to the vials walls during the process. To 100 µL of urine, 10 µL of internal standard (THCA-d3, 150 ng/mL in ACN) and 20 µL 9M NaOH were added, and the sample was incubated at 60°C for 20 minutes. Once cool, the sample was diluted with 200 µL ACN and neutralised with 10 µL glacial acetic acid. Final dilution with 200 µL 20 mM NH4
OAc
was performed to prepare the sample for the solid phase extraction procedure.
Oral fl uid was treated through mild protein precipitation prior to extraction. A combination or oral fl uid and preservation buffer (750 µL total volume containing 250 µL of oral fl uid) was treated with 200 µL ACN and 25 µL of internal standard solution (THCA-d3 at 1 ng/mL and THC-d3 at 10 ng/mL), followed by 50 µL of 1% NH4
OH solution.
O/ACN (50:50 v:v) before elution with ACN/Formic Acid (95:5 v:v) into Thermo Scientifc™ WebSeal™ Glass Inserted plates. Urine samples were eluted with two 50 µL aliquots, while oral fl uid samples were eluted with two 30 µL aliquots. Extracts were diluted 1:1 with H2
Treated samples were then loaded into the SOLAµ SAX well plate and drawn through under vacuum. Samples were washed with 200 µL of H2
O prior to injection.
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