50 February / March 2016


Figure 4. (a) Calibration curve from 0 to 1000 ng/mL for the LC-MS/ MS analysis of THC in oral fl uid following SPE. (b) Chromatogram of the lowest standard (THC = 0.5 ng/mL.


neutralisation and dilution, this introduces contaminants and potential interferences that can negatively impact the lifetime of the analytical column and the overall cleanliness and hence sensitivity, of the mass spectrometer. Solid phase extraction is an alternative that produces much cleaner samples. However, any method involving solid phase extraction should be as simple as possible to facilitate rapid sample preparation and reduce the potential for errors.


The procedure developed here involves base hydrolysis with concentrated NaOH followed by dilution and neutralisation. Analytes are then extracted from the hydrolysed sample followed by micro-elution solid phase extraction using a mixed-mode (reversed- phase/strong anion exchange) sorbent. This kind of material provides high selectivity for acidic analytes, resulting in cleaner extracts that eliminate more contaminants and


Chromatography and MS Detection


LC separations were performed using Thermo Scientifi c™ Accucore™ RP-MS analytical column (100 x 2.1 mm, 2.6 µm) with Mobile Phase A containing 0.1% formic acid in water and Mobile Phase B containing 0.1% formic acid in acetonitrile at a fl ow rate of 0.40 mL/min. An initial mobile phase composition of 60% B was held for 0.50 minutes before applying a linear gradient to 95% B for 3.0 minutes before a hold time of 0.50 minutes. The mobile phase was returned to the initial conditions for column equilibration, resulting in a total run time of 5.0 minutes. Mass data was acquired in SRM mode using the ion source conditions and MS/MS transitions listed in Table 1.


Results and Discussion THCA in Urine


When determining the amount of THCA in urine, samples should be hydrolysed with strong base to convert any THCA- glucuronide present into the uncomplexed form to simplify the analysis and data interpretation. Although it is possible to inject the hydrolysed samples following


interferences to produce better analytical column lifetimes and reduces intervals between instrument maintenance. Elution is performed using acetonitrile with 5% formic acid, generating a sample that can be injected into the LC-MS/MS system following dilution with water, eliminating the common evaporation and reconstitution steps while still meeting necessary detection limits.


Guidelines provided by the Substance Abuse and Mental Health Services Administration (SAMHSA) set a cutoff for a positive THCA determination in urine is 15 ng/mL [10]. To ensure that the method would meet requirements by having a limit of detection at or below 10% of the cutoff value, linearity was determined by spiking blank urine with THCA in concentrations from 0 to 100 ng/mL. The ratio of the peak area for THCA to the peak area for the internal standard (THCA-d3) was used to calculate the response for each sample. Linear response over the full range was observed with no weighting (R2


=0.9992)


(Figure 2a) with the lowest concentration standard (1.5 ng/mL) showing more than adequate signal-to-noise (Figure 2b). Although a true limit of detection was not determined for the method, this result


suggests that it is well below the 10% of cutoff threshold.


Recovery of THCA from the solid phase extraction process was assessed by comparing the THCA peak area for samples spiked with THCA before and after extraction for three different concentrations (1.5, 15, and 40 ng/mL). Six replicates were performed at each concentration level. At all three levels, recoveries greater than 92% were observed for THCA with high reproducibility indicated by %RSD less than 5.4%. Minimal matrix effects were observed by comparing post-extract spiked samples to samples prepared by spiking the same amount of THCA into the elution solvent.


Quality control samples were prepared by spiking blank urine with THCA at concentrations above and below the cutoff limit. These samples were extracted and analysed, and the calculated concentrations were compared to the actual concentrations. Six replicates were analysed at each concentration. For all samples, the calculated THCA concentration agreed with the actual concentration to within 5%.


THC and THCA in Oral Fluid


Oral fl uid is a challenging matrix to work with due to the generally lower concentrations of drugs and metabolites relative to other fl uids such as urine and blood [11]. For example, while analytical methods for determining THCA levels in urine need only to be suitable to concentrations around 1.5 ng/mL, methods for determining THCA in oral fl uid must be capable of detecting and quantifying at levels of around 10 pg/mL [12].


A variation on the sample preparation procedure described above for urine has been applied to oral fl uid. Sample pre- treatment has been modifi ed to be more appropriate to the different matrix, using dilution with acetonitrile to help with protein removal and solubilisation of THCA. Again, while it is possible to analyse oral fl uid following simple dilution, solid phase extraction is generally a better approach since the sample can be concentrated down through the extraction procedure. A similar solid phase extraction procedure using SOLAµ SAX with a mixed-mode reversed- phase/strong anion exchange sorbent was used, with elution using acetonitrile with 5% formic acid that can be diluted with water prior to injection.


Limits of quantifi cation (LOQs) were defi ned as the lowest concentrations that had back- calculated values within 20%, and RSD for fi ve QC replicates within 20%. Using these


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