Validation of a liquid chromatography-tandem mass spectrometry method for analyzing cannabinoids in oral fluid.

A liquid chromatography tandem mass spectrometry method was developed for quantifying ten cannabinoids in oral fluid (OF). This method utilizes OF collected by the Quantisal™ device and concurrently quantifies cannabinol (CBN), cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-THC (11-OH-THC), 11-nor-9-carboxy-Δ9-THC (THC-COOH), 11-nor-9-carboxy-Δ9-THC glucuronide (THC-COOH-gluc), Δ9-THC glucuronide (THC-gluc), cannabigerol (CBG), tetrahydrocannabiverin (THCV), and Δ9-tetrahydrocannabinolic acid A (THCA-A). Solid phase extraction was optimized using Oasis Prime HLB 30 mg 96-well plates. Cannabinoids were separated by liquid chromatography over a BEH C18 column and detected by a Waters TQ-S micro tandem mass spectrometer. The lower limits of quantification (LLOQ) were 0.4 ng/mL for CBN, CBD, THC, 11-OH-THC, THC-gluc, and THCV; and 1.0 ng/mL for THC-COOH, THC-COOH-gluc, CBG and THCA-A. Linear ranges extended to 2000 ng/mL for THC and 200 ng/mL for all other analytes. Inter-day analytical bias and imprecision at three levels of quality control (QC) was within ±15%. Mean extraction efficiencies ranged from 26.0-98.8%. Applicability of this method was tested using samples collected from individuals randomly assigned to smoke either a joint containing <0.1%, 5.9%, or 13.4% THC content. This method was able to identify and calculate the concentration of 6 of 10 cannabinoids validated in this method

Multicenter assessment of a hemoglobin A1c point-of-care device for diagnosis of diabetes mellitus.

ObjectiveA multisite investigation compared the analytical performance of a point-of-care (POC) HbA1c device with multiple commonly used HbA1c laboratory methods and an NGSP (National Glycohemoglobin Standardization Program) reference method.Research design and methodsThe Afinion AS100 POC device analyzed HbA1c using 618 EDTA whole blood excess patient specimens with clinically indicated HbA1c testing. Results were compared to measurements across five clinical laboratories and the NGSP reference method. Precision was evaluated over 8-10 consecutive days for low-, mid-, and high-range HbA1c specimens at all five sites.ResultsOver a wide range of HbA1c values (4.0%-15% HbA1c), 97.1% of the POC results and 94.5% of routine laboratory results fell within the target value of ±6% of the NGSP reference method results. The POC HbA1c results at 6.5% exhibited a total relative bias of -0.6% (-0.04% HbA1c) compared to the reference method while the aggregate of laboratory methods displayed a relative bias of -0.9% (-0.06% HbA1c). The total imprecision of the POC results ranged from 0.74-2.13% CV across the analytic measurement range compared to 0.81-3.23% CV for the routine laboratory methods.ConclusionsThe accuracy and precision of the Afinion POC HbA1c method was comparable to the laboratory HbA1c methods supporting the FDA's recent approval of the Afinion HbA1c Dx device for use in the diagnosis of diabetes

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 μg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving

Driving Under the Influence of Cannabis: Impact of Combining Toxicology Testing with Field Sobriety Tests

BACKGROUND: Cannabis is increasingly used both medically and recreationally. With widespread use, there is growing concern about how to identify cannabis-impaired drivers. METHODS: A placebo-controlled randomized double-blinded protocol was conducted to study the effects of cannabis on driving performance. One hundred ninety-one participants were randomized to smoke ad libitum a cannabis cigarette containing placebo or delta-9-tetrahydrocannabinol (THC) (5.9% or 13.4%). Blood, oral fluid (OF), and breath samples were collected along with longitudinal driving performance on a simulator (standard deviation of lateral position [SDLP] and car following [coherence]) over a 5-hour period. Law enforcement officers performed field sobriety tests (FSTs) to determine if participants were impaired. RESULTS: There was no relationship between THC concentrations measured in blood, OF, or breath and SDLP or coherence at any of the timepoints studied (P \u3e 0.05). FSTs were significant (P \u3c 0.05) for classifying participants into the THC group vs the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired at this same timepoint. Combining a 2 ng/mL THC cutoff in OF with positive findings on FSTs reduced the number of controls classified as impaired to zero, 86 minutes after smoking the placebo. CONCLUSIONS: Requiring a positive toxicology result in addition to the FST observations substantially improved the classification accuracy regarding possible driving under the influence of THC by decreasing the percentage of controls classified as impaired

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-β receptor (TPβ), but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPβ raised basal phosphorylated (p)FOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619). Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2) or ERK inhibitor (U0126). Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation

Effect of TP agonist on FOXO3 target proteins expressions and acetylation status.

<p>(<b>A</b>) UMUC3 cells treated with vehicle or 1 µM U46619 for 30, 120, and 240 minutes. Cell lysates were collected and analyzed by Western blot for total FOXO3 expression and downstream targets p27^Kip1, MnSOD, and Bim. (<b>B</b>) Protein expression of p300 and SIRT1 in same cell lysates. α-tubulin served as loading control. (<b>C</b>) Quantified values from blots of three independent experiments are graphed. (*) indicates p<0.05, One-way ANOVA, compared to time 0 for each Target ratio. (<b>D</b>) Increased SIRT1 expression mediated by U46619 in UMUC3 cells corresponded with decreased acetylated FOXO3. Total FOXO3 was immunoprecipitated from serum starved UMUC3 cells treated with vehicle or 1 µM U46619 for 30, 120, or 240 minutes. Eluted proteins were analyzed by Western blot with antibodies against acetylated lysine residues and FOXO3. Blots are representative of three independent experiments. Blots were quantified and ratios for (<b>E</b>) acetylated-lysine (acetyl-K) to FOXO3 were graphed. Significance was determined using repeated measures One-way ANOVA with Tukey post-hoc test. P<0.05.</p

Expression of mutant FOXO3^3A attenuated TP agonist mediated migration of UMUC3 cells.

<p>(<b>A</b>) GFP-FOXO3^3A or EGFP-C₂ (Vector) was stably transfected into UMUC3 cells and mutant FOXO3 expression was confirmed by immunoblotting for GFP. α-tubulin served as loading control. Cells were seeded into inserts coated with fibronectin for migration or matrigel for invasion in serum free media containing 1 µM U46619 and placed in wells containing complete media with 1 µM U46619. Cells were allowed to migrate (<b>B</b>) for 8 hours or invade (<b>C</b>) for 16 hours. Significance was determined with a two-sided two-sample <i>t</i>-test, (* = P<0.05, n = 3) compared to GFP-vector control. (<b>D</b>) UMUC3 cells were pretreated with 10 µM U0126 for 15 minutes before stimulation with 1 µM U46619 and allowed to migrate for 16 hours. Data represent three independent assays done in duplicates and expressed as the mean ± SEM. Significance was determined with a One-way ANOVA with Tukey post-hoc test. P<0.05.</p

Decreased FOXO3 transcriptional activity and nuclear localization following TP agonist treatment.

<p>(<b>A</b>) UMUC3 cells transiently transfected with 3× forkhead response element (FHRE) reporter construct and Renilla luciferase control vector were treated with 1 µM U46619 for 30 or 120 minutes and resulted in decreased FOXO3 transcriptional activity. The average Firefly luciferase expression was adjusted to total protein and control vector Renilla luciferase expression before normalization to vehicle control. Results represent the average percent luciferase activity of three independent experiments conducted in duplicates. *P<0.05, One-way ANOVA with Tukey post-hoc test. (<b>B</b>) UMUC3 cells were serum starved overnight and pretreated for 15 min. with 1 µM Indomethacin prior to treatment with either vehicle or 1 µM U46619 for 5, 30, or 120 minutes. The cells were subjected to nuclear and cytoplasmic fractionation and proteins were analyzed by Western blot. α-Tubulin and Lamin-B1 served as loading controls for cytoplasmic and nuclear fractions, respectively. (<b>C</b>) Densitometry of blots showing prolonged agonist stimulation increased cytoplasmic FOXO3 protein and increased nuclear pFOXO3 proteins compared to vehicle controls. Significance was determined for all quantified blots using a repeated measure One-way ANOVA with Tukey post-hoc test. P<0.05.</p