Triniti Scroggin (Presenter)
Authorship: Triniti Scroggin2, Fang Wu3, Torri D. Metz4,5, Amanda A. Allshouse, Ingrid Binswanger, Gwendolyn A. McMillin1,2
1Department of Pathology, University of Utah, Salt Lake City, UT 84132, 2 ARUP Laboratories, Salt Lake City, UT 84108, 3Department of Laboratory Medicine and Pathology Saskatoon Health Region & University of Saskatchewan, SK, Canada, S7W 0S1, 4Denver Health and Hospital Authority, Denver CO 80204, 5University of Colorado School of Medicine, Aurora, CO 80045
Detection of in utero exposure to drugs is critical to the immediate and long term medical and social management of newborns. Our objective was to develop an LC-MS/MS method for the detection and quantification of 5 cannabis analytes in the traditional neonate specimen type (meconium) and a relatively new specimen type (umbilical cord tissue) considering the wide usage of cannabis amongst pregnant women. This method could support studies designed to evaluate the patterns and concentrations of cannabis analytes observed in newborns exposed to cannabis in utero, as well as correlate such data with clinical and social outcomes.
Detection of in utero exposure to drugs is critical to the immediate and long term medical and social management of newborns. Studies have shown that approximately 5% of pregnant women admit to using cannabis sometime during pregnancy. Being a widely used drug amongst pregnant women, our objective was to develop an LC-MS/MS method for the detection and quantification of 5 cannabis analytes in the traditional neonate specimen type (meconium) and a relatively new specimen type (umbilical cord tissue). This method could support studies designed to evaluate the patterns and concentrations of cannabis analytes observed in newborns exposed to cannabis in utero, as well as correlate such data with clinical and social outcomes.
Sample preparation methods were developed for detection and quantification of Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-Δ9-THC (THCA), 11-hydroxy-Δ9-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD) in umbilical cord tissue and meconium. Residual clinical samples were collected and deidentified according to Institutional policies for use in this study. Samples were weighed and homogenized. Supernatant was subjected to hydrolysis, solid phase extraction (SPE), concentration, and reconstitution. Purified extracts were analyzed on an AB SCIEX 5500 mass spectrometer interfaced with CTC PAL HTC-xt DLW autosampler and Agilent 1260 infinity series HPLC pump. Liquid chromatography (LC) separation was performed on an Agilent Poroshell 120 EC-C18 column, 3.0x50mm, 2.7µm column with mobile phase A: 5mM ammonium bicarbonate pH 9.5 in water and mobile phase B: methanol. 50µL of extracted sample were injected to the column at 28° C while a 750 µL/mL flow rate was applied for a total run time of 4.7 minutes. A LC profile was developed with an isocratic hold at 60% B for 0.5 minutes, from 0.5-1.2 minutes conditions were increased from 60-80% B, a second isocratic hold from 1.2-2.3 minutes at 80% B followed by a gradient increased from 2.3-3.2 minutes from 80-95% B, conditions were held at 95% B for 1.1 minutes, equilibration at 60% B was established from 4.1-4.7 minutes. LC eluent was diverted to the mass spectrometer from 0.8-4.3 minutes. Detection was performed by tandem mass spectrometry (MS/MS) in negative electrospray ionization (ESI) mode. Optimized parameters: collision gas 10 psi, curtain gas 30 psi, ionspray voltage -4000v, source temperature 550° C, nebulizer gas 25 psi, and heater gas 30 psi. Two transitions were monitored for each standard and its respective internal standard (deuterated analogs). Data was acquired by multiple reaction monitoring (MRM) mode by AB SCIEX Analyst software. Analysis was completed in AB SCIEX MultiQuant software using MQ4 algorithm and linear regression (1/x weighing). Calibration curves were prepared with drug-free matrix. Validation included linearity, imprecision, sensitivity, recovery, and patient correlation studies. Assay performance criteria were correlation coefficient over the analytical measurement range of R2≥0.995, average imprecision (% coefficient of variation) ≤20%, limit of detection S/N (signal to noise ratio) ≥3, limit of quantitation S/N≥10 and correlation/accuracy ≥80%.
Two sources of authentic specimens were studied. First, seven paired meconium and cord samples (collected from the same birth), for which meconium was positive for THCA using a previously validated method, were analyzed to compare concentrations and patterns of analytes between the specimen types. The second sample set was umbilical cord tissue collected as part of a cross-sectional study of pregnant women delivering at two urban Colorado hospitals that included self-report of cannabis use patterns by anonymous survey, approved by the Colorado Multiple Institutional Review Board (n=116).
Method Validation: Limit of quantitation and limit of detection were 0.2 ng/g and 0.1 ng/g for all 5 cannabinoids. Validation criteria were met for both umbilical cord and meconium assays with the exception of CBD. Final extract recovery for all analytes but CBD in umbilical cord and meconium ranged from 71-99% and 73-83%, respectively. Average CBD recovery was poor; less than 15% in cord and 27% in meconium, so CBD was excluded from analyses.
Sample set 1: Paired samples illustrated analyte patterns and concentration differences based on sample type. THCA was detected in all 7 sets of paired specimens. Concentrations in cord were consistently lower when compared to meconium for all analytes: 0.138-8.71 ng/g for umbilical cord and 2.4-537 ng/g for meconium. In umbilical cord THCA, THC, CBN, and 11-OH-THC were present in 100% (n=7), 71% (n=5), 57% (n=4), and 57% (n=4) of the paired samples, respectively. In meconium THCA, THC, CBN, and 11-OH-THC were present in 100% (n=7), 100% (n=7), 100% (n=7), and 43% (n=3) respectively.
Sample set 2: Of the University of Colorado umbilical cord samples, 27.6% (n=32) were positive one or more of the 5 cannabinoids (cutoff 0.1 ng/g). THCA was observed in all (n=26) but six of the positive cord samples; non-THCA analytes were observed in 14 of the cords. Of note, four samples were positive for only 11-OH-THC and one sample was positive for only THC. Considering maternal self-reported use of cannabis within 30 days of delivery 100% (n=6) were positive for THCA, whereas 18% (20/110) were positive for cannabis analytes among women without self-reported use in the 30 days prior to delivery (p<0.001).
Collectively, THCA was the most frequently observed analyte seen in the authentic umbilical cord samples analyzed (n=33 out of 123: 7 paired samples and 116 self-report samples). Of the 123 samples 11-OH-THC and THC were present in 11.4% (n=14) and 11.4% (n=14) of the samples, respectively. The least common analyte being CBN seen in 6.5% (n=8) of the samples. Of note, 5% (n=6) of the positive samples did not contain THCA.
Conclusions & Discussion
A LC-MS/MS method was developed and validated to detect and quantify 4 cannabinoids in both umbilical cord and meconium. Concentrations and patterns of cannabis analytes varied considerably between umbilical cord and meconium samples. Concentration of analytes in umbilical cord tissue were an order of magnitude lower than in meconium, suggesting the need for very different cutoff concentrations to minimize false negative results. Analytes other than THCA were commonly observed, but the relevance of the patterns and concentrations of the individual analytes is not yet understood. Sensitivity of 0.1 ng/g is appropriate based on a limited population of umbilical cord specimen with maternal self-reported cannabis use within 30 days of delivery. However, a large number of cord specimen from maternal self-reported cannabis use might be necessary to achieve confident conclusions. Moreover, a number of cord samples from births that were not associated with self-reported cannabis use were also positive suggesting that studies will be required to evaluate clinical specificity of this cutoff concentration, as well as the value of quantitative results. In summary, the assay described here could help clinicians and researchers answer those and other questions by evaluating clinical significance of concentrations and analyte patterns in umbilical cord tissue and meconium collected from cannabis-exposed newborns.
References & Acknowledgements:
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