Highlights
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A LC-MS/MS method was developed for the detection and quantification of 5 cannabinoids.
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Paired meconium and umbilical cord samples were analyzed to determine in utero exposure.
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Concentrations and analyte profiles varied between umbilical cord tissue and meconium samples.
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Our results suggest the need for matrix dependent cutoffs to minimize false negative results.
Abbreviations: CBN, cannabinol; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; NaOH, sodium hydroxide; NH4OH, ammonium hydroxide; RT, retention time; THC, Δ9-tetrahydrocannabinal; THCA, 11-nor-9-carboxy-THC (THCA); 11-OH-THC, 11-hydroxy-THC (11-OH-THC
Keywords: In utero drug exposure, Cannabinoids, Meconium, Umbilical cord tissue, LC-MS/MS
Abstract
Understanding levels of in utero drug exposure is important to properly customize the immediate, as well as ongoing, medical and social management needs of affected newborns. Here, we present the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of 4 cannabinoid analytes in two neonatal matrices. The analytes targeted were Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN). The matrices analyzed were umbilical cord tissue and meconium. A fifth analyte, cannabidiol (CBD), was also detected uniquely in meconium. Extracts were analyzed by LC-MS/MS in negative electrospray ionization mode.
Paired meconium and umbilical cord samples (i.e., one specimen from each matrix collected from each single birth, n = 46 pairs) were tested to evaluate concentration and metabolite profiles. THCA was detected in all positive (containing one or more analytes) meconium samples (n = 32). CBN, THC, 11-OH-THC, and CBD were present in 57% (n = 26), 39% (n = 18), 24% (n = 11), and 20% (n = 9), respectively. Concentrations were lower in the umbilical cord samples for all analytes (i.e., 0.27–537 ng/g for meconium and 0.1–9 ng/g for umbilical cord). In umbilical cord THCA was also detected in all positive samples (n = 19) while THC, CBN, and 11-OH-THC were present in 24% (n = 11), 17% (n = 8), and 11% (n = 5), respectively. Testing neonatal matrices for cannabinoids could be used to support studies designed to detect newborns exposed to cannabis in utero, as well as provide data that could be examined for correlations with clinical and social outcomes.
1. Introduction
Cannabis (e.g., marijuana) is widely used in the United States, where it is now legal for either medicinal or recreational use in a majority of states, but remains illegal based on federal scheduling. The national survey of drug use and health report from 2016 showed an increase of past marijuana use among all individuals over 12 years of age [1]. Increased admission of marijuana use was also noted in pregnant women. For example, approximately 8.5% of pregnant women aged 18–25 admitted to the use of marijuana during the past month in the 2016 survey, compared to 6.4% in 2015 [1]. The increased prevalence of cannabis use in pregnant women has raised concerns about the impact of cannabis exposure to the fetus, and potential outcomes.
The cannabis leaf contains more than 100 cannabinoids and over 500 other chemicals, with THC being the major psychoactive phytocannabinoid that has been identified [2]. THC is commonly used for its intoxicating effects, including euphoria and sedation. During pregnancy, cannabis has been used to decrease nausea, pain, anxiety, insomnia, and to stimulate appetite. A study from Vancouver, Canada found that up to 77% of medicinal cannabis use during pregnancy was related to nausea and over 50% reported cannabis use to treat lack of appetite, general pain, insomnia, anxiety, depression and fatigue [3]. Evidence shows that cannabinoids freely cross the placenta and directly reach the fetus [4], [5], [6]. Cannabinoids act on the endocannabinoid system that is important to the development of the embryonic brain from early neural stem cell survival, to the differentiation of neuronal cells, and synaptic function [7], [8], [9]. During early stages of brain development, cannabinoids may affect sensory input, control of body temperature, and higher order cognitive processes including memory, learning, anxiety, depression, pain, and decision making [10], [11]. The Ottawa prenatal prospective study (approximately n = 180) annually monitored individuals that were exposed to cannabis in utero until the age of 6 and then less frequently thereafter until the age of 9. Despite several limitations of the study design, results suggest behavioral problems, reduced performance on visual perception tasks, language comprehension issues, sustained attention deficiencies, and memory difficulties at 6–9 years of age that may be related to pre-natal cannabis exposure [12].
Testing of neonate specimens for the presence of drugs and drug metabolites, as well as their concentration profiles, can provide convincing evidence of pre-natal drug exposure and a better understanding of thresholds that can predict newborns who are at risk of future medical and social complications [13], [14], [15], [16]. That said, positive results do not indicate the timeframe, level, or duration of drug exposure, nor does a negative result rule out in utero exposure. Analysis of meconium has been used to assess in utero exposure to drugs, including cannabinoids, for more than 20 years [17], [18], [19], [20]. Meconium is the first stool passed by the newborn, and it begins to form and collect in the intestine between the 12th and 16th week of gestation. Collection of meconium is unpredictable due to retention for several days after birth or passage prior to, or during, delivery. Meconium is preferred over hair, urine, amniotic fluid, and blood due to the broad time window to exposure it provides that reflects approximately the last trimester of a full term birth [21], [22], [23], [24]. In recent years, umbilical cord tissue has become an alternative matrix [24]. The umbilical cord is the life line between the fetus and mother and is formed in approximately the 5th to 6th week of gestation. Although not as well understood as a site for drug deposition, analysis of umbilical cord has proven to be sensitive to exposure for many drugs over the last trimester, and exhibits several advantages over meconium (e.g., ease of collection at birth, and ample sample volume). Moreover, umbilical cord testing can avoid misleading results caused by administration of medications to the newborn directly after birth, prior to collection of meconium.
Traditional algorithms for drug testing include screening, often by immunoassay, followed by confirmation testing, typically performed by a chromatographic method coupled to mass spectrometric detection. Our laboratory has previously evaluated methods for detection of exposure to cannabis based on targeting a single analyte [25], [19], and proposed that a direct targeted approach to drug testing would be more efficient, and potentially more sensitive, than the traditional screen-with-reflex approach [26]. Here, we described a method designed to detect and quantify 4 cannabinoids in both the meconium and umbilical cord samples. The cannabinoids targeted include Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN). Cannabidiol (CBD) was detected uniquely in meconium. In addition, 46 paired authentic clinical samples (both meconium and umbilical cord collected from a single birth) were analyzed to assess the similarities and differences in metabolite concentrations and abundance profiles between matrices from the same birth. In addition to identifying in utero exposure to cannabis, data produced from this method could be correlated with clinical and social outcomes to help construct prognostic flow charts to assist in identifying if, and perhaps even when, an individual is likely to need clinical and/or social support in the future.
2. Methods and materials
2.1. Chemicals and reagents
Ethyl acetate, hexane, methanol and acetonitrile were HPLC grade and purchased from VWR international (West Chester, PA, USA). Clinical laboratory reagent water was generated using a Milli-Q integrated water system by Millipore Sigma (Burlington, MA, USA). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): glacial acetic acid, ammonium bicarbonate, ammonium hydroxide (NH4OH), and sodium hydroxide (NaOH). Stock standards and matched deuterated standards for all analytes were purchased from Cerilliant (Round Rock, Texas, USA).
2.2. Preparation of meconium calibration standards and quality control (QC) solutions
A primary Cerilliant stock solution containing THC, 11-OH-THC, THCA, CBN, and CBD, each at 100 µg/mL, was prepared in methanol. The stock solution was diluted to create four calibration solutions at 10 µg/mL, 6.5 µg/mL, 3.5 µg/mL, and 50 ng/mL. A calibration curve was generated by fortifying blank (previously verified by an existing ELISA method to be free of cannabinoids) meconium with stock solutions to create matrix-matched calibrators with the following concentration: 5, 350, 650, and 1000 ng/g. QC stock solutions were prepared independently of the calibration standards and included one high control where THCA-glucuronide was added as an indicator of hydrolysis and the free drug was excluded. QC solutions were used to fortify blank meconium for a final concentration of 7 and 750 ng/g, respectively. Primary stock solutions of THC-d3, THCA-d3, 11-OH-THC-d3, CBN-d3, and CBD-d3 were diluted in methanol to produce a mixed internal standard solution of 5000 ng/mL. All stock and working standards were aliquoted and stored in −80 °C in amber glass vials.
2.3. Preparation of umbilical cord calibration standards and quality control (QC) solutions
A primary Cerilliant stock solution containing THC, 11-OH-THC, THCA, and CBN at 50 µg/mL was prepared in methanol. The stock solution was diluted to create four calibration solutions at 400, 200, 20, and 8 ng/mL. A calibration curve was generated by fortifying blank pooled umbilical cord (previously verified to be free of cannabinoids) with stock solutions to create matrix matched calibrators with the following concentration 0.2, 0.5, 5.0, and 10.0 ng/g. QC stock solutions were prepared independently of the calibration standard and included one high control where THCA-glucuronide was added as an indicator of hydrolysis and the free drug was excluded. QC solutions were used to fortify blank umbilical cord for a final concentration of 0.25 and 7.5 ng/g, respectively. Primary stock solutions of THC-d3, THCA-d3, 11-OH-THC-d3, and CBN-d3 were diluted in methanol to produce a mixed internal standard solution of 100 ng/mL. All stock and working standards were aliquoted and stored in −80 °C in amber glass vials.
2.4. Sample extraction procedure: Meconium
Blank meconium samples, for the calibrators and controls, were weighed out (0.25 ± 0.01 g) into 2 mL Eppendorf polypropylene tubes. Patient samples were weighed out (0.25 ± 0.01 g) into pre-labeled 2 mL tubes. All patient samples and QC samples were spiked with 25 µL of the deuterated internal standard solution and 1.3 mL of methanol. QC and calibrator samples were spiked with 25 µL of the appropriate stock solution and all samples received ½ scoop of 0.5 mm stainless steel beads (Next Advance, NY, USA). Tubes were capped and samples were homogenized using a Bead Rupter (Biotage, NC, USA) for 4 cycles, each 10 s long, at speed 3.1 with a dwell time of 10 s. Homogenates were centrifuged for 10 min at 14,000 rpm (20,598×g) and 0–4 °C. Each supernatant was transferred to a 10 mL culture tube. Base hydrolysis was performed by adding 1.3 mL 0.5 mol/L NaOH. Basic supernatants were incubated at ambient temperature (23–25 °C) and continuously shaken for 20 min on a VWR advanced multi-tube vortexer at 230 V with a speed 700 rpm. Following hydrolysis, solid phase extraction was performed using Evolute AX SPE columns (60 mg/3 mL, Biotage). SPE sorbents were washed and equilibrated with 1 mL methanol and 1 mL water using a positive pressure manifold (SPEware Incorporated, San Pedro, CA) applying pressure to achieve a 1 drop/3 s rate. After conditioning, calibrators, controls, and patient specimens were loaded by gravity onto the SPE columns. If samples did not load by gravity pressure was applied to achieve a 1 drop/5 s rate. SPE sorbent were washed with 1 mL 1% NH4OH in 85:15 water: acetonitrile followed by 1 mL 50:50 hexane: ethyl acetate at 1 drop/s. Analytes were eluted into silanized conical bottom LC vials with two 600 µL fractions of 2% acetic acid in 90:10 hexane: ethyl acetate. Samples were concentrated at 40 °C under a gentle nitrogen stream at 30 psi with a Turbo Vap LV (Biotage, Charlotte, NC, USA) for approximately 15 min. Reconstitution was completed by the addition of 150 µL of water: methanol (40:60) and autosampler vials were capped with pre-slit cap prior to instrument analysis.
2.5. Sample extraction procedure: Umbilical cord
Sample extraction procedures followed for umbilical cord were previously published with minor changes described here, including sample pre-treatment, homogenization, and hydrolysis changes [27]. Blank umbilical cord samples were weighed (1.0 ± 0.1 g) for the calibrators and controls into 7 mL Eppendorf polypropylene tubes with a screw top. Following the calibrator and control weigh out, patient samples were placed in a weigh boat and cut into ½ cm cubes with a disposable razor blade and 1.0 ± 0.1 g was weighed into pre-labeled 7 mL tubes. All patient samples and QC samples were spiked with 25 µL of the deuterated internal standard solution and 2.5 mL of methanol. QC and calibrator samples were spiked with 25 µL of the appropriate stock solution and all samples received six 5.6 mm UFO stainless steel beads (Next Advance, NY, USA). Tubes were capped and samples were placed in a −70–80 °C freezer for 10 min prior to homogenization. Samples were homogenized using a Bead Rupter (Biotage, NC, USA) for 4 cycles, each 30 s long, at speed 5.0 with a dwell time of 30 s. Homogenates were centrifuged for 10 min at 14,000 rpm (20,598×g) and 0–4 °C. Supernatants were transferred to 10 mL culture tubes; base hydrolysis was performed in the same manner as meconium but with 2.5 mL 0.5 mol/L NaOH. Basic supernatants were incubated at ambient temperature (23–25 °C) and continuously shaken for 20 min on a VWR advanced multi-tube vortexer, 230 V at speed 700 rpm. Following hydrolysis, solid phase extraction was performed using Evolute AX SPE columns (60 mg/3mL, Biotage), as described previously (28).
2.6. LC-MS/MS conditions for meconium and umbilical cord
Chromatography, analysis parameters and associated instrumentation were identical for both the extracted meconium and umbilical cord samples. LC separation was performed on a Poroshell 120 EC-C18 column (2.1 × 50 mm, 2.7 µm particle size) paired with an in-line filter (Agilent, Santa Clara, CA). Solvent A (5 mM NH4OH in nanopure water, pH 9.5) and solvent B (methanol) were used to develop a gradient: 60% B for 0.50 min, linear gradient from 60% B to 80% B between 0.50 and 1.20 min, hold at 80% B for 1.10 min, and a linear gradient from 80% B to 95% B between 2.30 and 3.20 min, and hold at 95% B for 50 s followed by re-equilibration to initial condition (total run time 4.7 min). The LC eluent was diverted to waste for the first 0.5 min and the final 0.5 min. LC flow rate was 0.75 mL/min with an injection volume of 5 µL for meconium and 50 µL for umbilical cord (Fig. 1 for chromatographic separation). The column and autosampler were held at 28 °C and 4 °C, respectively. MS/MS analysis was performed by AB SCIEX Triple Quad™ 5500 mass spectrometer interfaced with CTC PAL HTC-xt-DLW autosampler and Agilent 1260 infinity series binary pump, degasser and column oven. Samples were ionized in negative electrospray mode and data were collected using multiple reaction monitoring, see parameters in Table 1. MS/MS parameters were optimized via direct infusion of a mixture of standards followed by a split tee injection with LC flow (curtain gas 30 psi, collision gas 10 psi, ion spray voltage −3500 v, 600 °C, nebulizer gas 20 psi, and heater gas 30 psi). AB SCIEX Analyst software (version 1.6.1) was used for instrument control. Two transition ions were monitored for each drug analyte and its respective deuterated standard, as shown in Table 1. Ion ratios were calculated by dividing the peak area of the qualifier ion by the peak area of the quantifier ion. An average ion ratio was calculated using the data obtained with calibrators and controls; ion ratios of positive patient samples were monitored and were considered acceptable if results fell within ±30% of the established average. Quantitation was performed using MultiQuant™ 3.0.2 software (AB Sciex, Foster City, CA) integrating with the MQ4 algorithm. Calibration curves were constructed via linear least-squares regression with 1/x weighting factor.
Fig. 1.
Representative LC separation and extracted ion chromatograms of 1) 11-nor-9-carboxy-THC (THCA), 2) 11-hydroxy-THC (11-OH-THC), 3) cannabidiol (CBD), 4) cannabinol (CBN), 5) Δ9-tetrahydrocannabinal (THC) and matched (deuterated d3) internal standards at the limit of quantitation for meconium (5 ng/g) and umbilical cord (0.2 ng/g).
Table 1.
Multiple reaction monitoring transitions (MRM) for non-deuterated and internal standards for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD) with the matched deuterated (d3) internal standards and their respective retention times (RT).
| Non-deuterated analyte/deuterated internal standard | RT (min) | MRM transitions for non-deuterated analyte | MRM transitions for deuterated analyte |
|---|---|---|---|
| THC/THC-d3 | 3.8 | 313.10 → 245.1 313.10 → 191.1 |
316.0 → 248.0 316.0 → 194.0 |
| THCA/THCA-d3 | 1.9 | 343.1 → 244.9 343.1 → 191.0 |
346.0 → 248.2 346.0 → 194.0 |
| 11-OH-THC/11-OH-THC-d3 | 2.9 | 329.1 → 268.1 329.1 → 173.3 |
332.1 → 271.0 332.1 → 173.0 |
| CBN/CBN-d3 | 3.6 | 309.3 → 222.1 309.3 → 171.0 |
312.0 → 282.0 312.0 → 171.0 |
| CBD/CBD-d3 | 3.1 | 313.0 → 245.1 313.1 → 179.0 |
316.1 → 248.0 316.1 → 182.0 |
2.7. Method validation
Validation experiments included within- and between-run imprecision, accuracy, patient correlation, linearity, sensitivity, SPE recovery, method recovery, matrix effect, carryover and on-board stability of extracts using blank meconium and umbilical cord fortified with non-deuterated and deuterated standards.
Within-run, between-run and total imprecisions of the assay (percent coefficient of variation, %CV) were evaluated by extracting samples across the analytical measurement range (AMR) in triplicate over five days. Accuracy was determined using calibrators (prepared in matrix) tested in triplicate over five days where the experimental concentrations were compared to the expected concentration. The linearity of the assay was assessed by evaluating the data generated by a four-point calibration curve run in triplicate for 3 days alongside 5 AMR samples. Samples were prepared with drug-free meconium and umbilical cord fortified with an appropriate concentration of the analytes. The lower limit of quantitation (LLOQ) was defined as the lowest calibrator, and the validation was designed to evaluate performance with a signal to noise ratio (S/N) ≥ 10, as well as all qualitative and quantitative parameters, including Gaussian chromatography, retention time (RT) within ±2% of the target RT, all monitored ions present and all ion mass ratios (calculated by dividing the area of quantifier ions by the area of qualifier ions) within ±30% of the mean ion mass ratios of calibrators and controls, accuracy within 85–115% of the target concentrations and imprecision ≤20%. The limit of detection (LOD) for each analyte was not specifically determined, but performance of the method at concentrations equivalent to half of the LLOQ was verified to ensure that the acceptance criteria defined above and a S/N of ≥3 were met for each analyte. The upper limit of quantitation (ULOQ) was the highest concentration at which all samples were accurate within 85–115% of the target concentrations with imprecision ≤20%. Solid phase extraction (SPE) recovery was evaluated by analyzing the same patient samples in triplicate over five days. Deuterated standard was added to a set of patient samples before and after SPE extraction. Patient sample recoveries were calculated based on the difference in concentrations between the pre-extracted and post-extracted samples. Method (analytical) recovery was determined by spiking a set of drug-free samples with a known amount of standard. Method recovery was evaluated by how well the method accurately quantitated the spike samples where the expected and experimental concentrations were compared. Patient correlation was determined by analyzing 40 samples previously analyzed by a validated method along with 30 fortified negative patient samples. Hydrolysis efficiency was calculated for THCA-glucuronide using the high control spiked 1.51 times the target concentration due to mass differences between glucuronidated and free drug. Mass spectrometry ion suppression was evaluated by injecting negative meconium or umbilical cord extracts while infusing a solution containing each analyte of interest at 250 and 5 ng/g, respectively. Carryover studies were performed to assess a) carryover due to a contaminated syringe, and b) septum/injection port/column carryover using samples prepared at concentrations twice the ULOQ followed by samples prepared with concentrations near the low end of the AMR (i.e., 7 ng/g). Internal standard counts were monitored within runs and between runs to assure consistency across a run (±30%). On-board stability of the extracted analytes was evaluated by re-analysis of a full batch (n = 54) every 24 h for 3 days. The concentration and area counts at each time point were compared to the initial condition. The ratios of the results were calculated. EP Evaluator software (Data Innovations, Burlington, USA), Multiquant, and Excel (Microsoft, Redmond, WA) were used for data analysis.
2.8. Authentic clinical specimens
Forty-six paired meconium and umbilical cord samples collected from the same birth were evaluated with this method. Samples were selected for inclusion in this study based on the ordering patterns for archived clinical specimens [28], [29]. Thus, specimens for which both meconium and umbilical cord testing for THC was requested for the same newborn were sought. Following procedures approved by the Institutional Review Board of the University of Utah, residual paired samples (indifferent of THC results) were retrieved prior to scheduled discard dates. The specimens were de-identified, and analyzed with the method described here. The concentrations of five cannabinoids in meconium and four cannabinoids in umbilical cord in authentic specimens were analyzed by the same LC-MS/MS method described above and metabolite profiles, as well as respective concentrations, were assessed. Data was analyzed in MultiQuant™ and imported into Excel Spreadsheets (Microsoft, Redmond, WA), EP Evaluator, and Rstudio using ggplot2 [30] to perform statistical analysis.
3. Results and discussion
In this study, we present a method for the detection and quantitation of THC, THCA, 11-OH-THC, CBN and CBD in meconium and a similar method optimized for umbilical cord to detect and quantitate THC, THCA, 11-OH-THC, and CBN. The additional analytes could improve the sensitivity of analysis for in utero exposure to cannabinoids, particularly when mothers are using CBD-containing products instead of THC-containing products, and help support a better understanding of the abundance profiles of cannabinoids in meconium and umbilical cord.
Table 2, Table 3 detail the results for meconium and umbilical cord, respectively, for accuracy, within- and between-run imprecision, and extraction recoveries during validation. For meconium within-run (n = 15) and between-run (n = 15) imprecision at low control (n = 30, 7 ng/g) ranged from 1 to 3% and 4–6%, respectively, while, at high control (n = 30, 750 ng/g), within- and between-run imprecisions ranged from 2 to 3% and 3–13%, respectively. For umbilical cord within-run (n = 15) and between-run (n = 15) imprecision at low control (n = 30, 0.25 ng/g) ranged from 2 to 3% and 8–20%, respectively, while, at high control (n = 30, 7.5 ng/g), within- and between-run imprecision ranged from 4 to 6% and 8–15%, respectively. The average meconium SPE recoveries observed for THC, THCA, 11-OH-THC, CBN, and CBD were 73%, 83%, 74%, 83%, and 27%, respectively. The average umbilical cord SPE recoveries observed for THC, THCA, 11-OH-THC, and CBN were 74%, 82%, 58%, and 86%, respectively. Because minimal amounts of CBD could be recovered from umbilical cord, this analyte was excluded from the validated method. Using THCA-glucuronide as a hydrolysis indicator showed that base hydrolysis was effective to achieve concentrations within ±20% of the target concentration for both matrices. Short-term on-board stabilities of extracted THC, THCA, 11-OH-THC, CBN, and CBD were evaluated using 3 concentrations for meconium extracts and 7 concentrations for umbilical cord extracts. Expected sample concentrations were within ± 20% of the target value with CVs < 20% when maintained at 4 °C in the autosampler for 72 h. The analytical measurement range was 5–1000 ng/g for THC, THCA, 11-OH-THC, CBN, and CBD in meconium and 0.2–10 ng/g for THC, THCA, 11-OH-THC, and CBN in umbilical cord with r ≥ 0.995. No significant carryover was observed immediately after a sample containing 2000 ng/g of each analyte. Matrix effects in meconium were minimal for THC, THCA, 11-OH-THC, CBN, and CBD at 78%, 101%, 107%, 109%, and 79%, respectively (a value of 100% indicated no matrix effect). Matrix effects were observed in umbilical cord matrices with 93% (THC), 91% (THCA), 94% (11-OH-THC), and 92% (CBN), respectively.
Table 2.
Validation results for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD) in meconium by LC-MS/MS.
| Analyte* | Average Accuracy (N = 36) | Imprecision (%Coefficient of Variation) |
Recovery |
||||||
|---|---|---|---|---|---|---|---|---|---|
| Low (N=15) 7.5 ng/g |
High (N=15) 750 ng/g |
Method | Solid Phase Extraction | ||||||
| Total | Between Run | Within Run | Total | Between Run | Within Run | ||||
| THC | 99% | 5% | 5% | 1% | 3% | 3% | 2% | 86% | 73% |
| THCA | 98% | 5% | 4% | 2% | 7% | 7% | 2% | 102% | 83% |
| 11-OH-THC | 98% | 6% | 6% | 2% | 4% | 4% | 2% | 85% | 74% |
| CBN | 97% | 4% | 4% | 3% | 7% | 6% | 3% | 83% | 83% |
| CBD | 97% | 6% | 5% | 3% | 13% | 13% | 2% | 94% | 27% |
Limit of quantitation and the limit of detection for all analytes in meconium is 5 ng/mL and 2.5 ng/mL, respectively.
Table 3.
Validation results for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), and cannabinol (CBN) in umbilical cord by LC-MS/MS.
| Analyte* | Average Accuracy (N = 36) | Imprecision (%Coefficient of Variation) |
Recovery |
||||||
|---|---|---|---|---|---|---|---|---|---|
| Low (N = 15) 0.25 ng/g |
High (N = 15) 7.5 ng/g |
Method | Solid Phase Extraction | ||||||
| Total | Between Run | Within Run | Total | Between Run | Within Run | ||||
| THC | 97% | 14% | 14% | 2% | 9% | 8% | 4% | 99% | 74% |
| THCA | 98% | 8% | 8% | 3% | 12% | 10% | 6% | 97% | 82% |
| 11-OH-THC | 94% | 19% | 19% | 2% | 10% | 9% | 4% | 89% | 58% |
| CBN | 93% | 20% | 20% | 3% | 16% | 15% | 5% | 94% | 86% |
Limit of quantitation and the limit of detection for all analytes in meconium is 0.2 ng/mL and 0.1 ng/mL, respectively.
A set of 46 paired samples collected from the same birth were analyzed. Fig. 2 summarizes the concentrations of each analyte in both matrices. Meconium: 70% (n = 32) of samples were confirmed positive for one or more analytes. THCA was the most commonly observed analyte in meconium, present in 100% of the positive samples (n = 32) with concentrations between 1.4 and 537 ng/g (median = 21 ng/g). CBN was present in 57% of the samples (n = 26) with a concentration range of 0.27–46 ng/g (median = 6 ng/g). THC was present in 39% of the samples (n = 18) with a concentration range of 0.31–8 ng/g (median = 2 ng/g). 11-OH-THC and CBD were the least frequently identified analytes, being observed in only 24% (n = 11), and 20% (n = 9) of the samples with concentration ranges of 1.4–4 ng/g (median = 3) and 2–53 ng/g (median = 4), respectively. Umbilical cord: concentrations observed in umbilical cord were an order of magnitude lower than those in meconium. Similar to meconium, THCA was the most frequently observed analyte in umbilical cord, showing up in 41% (n = 19) of the samples with concentrations ranging from 0.1 to 9 ng/g (median = 2 ng/g). Interestingly, the second most frequently identified analyte in umbilical cord was THC, present in 24% (n = 11) of samples with a concentration range of 0.1–1 ng/g (median = 0.23 ng/g). CBN was present in 17% (n = 8) of the samples (range = 0.1–0.15 ng/g, median = 0.14 ng/g) followed by 11-OH-THC in 11% (n = 5) of the positive (for one or more analytes) samples (range = 0.2–0.33, median = 0.28 ng/g). These results highlight the discrepancies of cannabinoid concentrations between meconium and umbilical cord. Considering that little is known about the stability, drug deposition characteristics, and pharmacokinetics of each individual drug and its metabolites in both matrices, these discrepancies are not surprising. Fig. 3 shows the positivity rates of the five cannabinoids for the paired sample from the same birth. Again, discrepant positivity rates were observed between the two matrices. Of the 46 paired specimens, 32 meconium specimens were positive for one or more analytes, compared to 19 umbilical cord specimens positive for one or more analytes. This discrepancy suggests 13 false-negative umbilical cord results (a 40% false-positive rate). Upon qualitative assessment, the negative agreement between meconium and umbilical cord was 100%, while positive agreement was 59% between matrices. This anomaly suggests meconium is a more sensitive matrix for the detection of in utero exposure to cannabis, likely due to a less efficient deposition of cannabinoids in the umbilical cord. Further studies will be needed to correlate these metabolite profiles with the risk of adverse outcome. Raw patient data collected by the presented method are shown in the Table 4. Further data analysis showed that, of the 13 uniquely positive meconium samples (correlating negative umbilical cord results), 7 samples only contained THCA, while 3 contained all analytes except CBD. Only one sample contained all five cannabinoid analytes. A wide range of concentrations were detected in these 13 meconium samples, which overlapped with the range of concentrations observed in samples, wherein both meconium and umbilical cord results were positive. The wide range of concentrations may reflect differences in metabolism, cannabis use/exposure patterns, or physiological composition (e.g., chemical composition) of the specimens themselves.
Fig. 2.
Concentration distribution (log scale) of results positive for 11-hydroxy-THC (11-OH-THC), cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinal (THC), and 11-nor-9-carboxy-THC (THCA) in meconium and umbilical cord paired samples from the same birth.
Fig. 3.
Positivity rates of 11-hydroxy-THC (11-OH-THC), cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinal (THC), and 11-nor-9-carboxy-THC (THCA) for paired meconium and umbilical cord samples from the same birth.
Table 4.
Raw patient data for Δ9-tetrahydrocannabinal (THC), 11-nor-9-carboxy-THC (THCA), 11-hydroxy-THC (11-OH-THC), cannabinol (CBN), and cannabidiol (CBD) collected by LC-MS/MS.
| Meconium |
Umbilical Cord |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| THCA (ng/g) | THC (ng/g) | 11-OH-THC (ng/g) | CBN (ng/g) | CBD (ng/g) | THCA (ng/g) | THC (ng/g) | 11-OH-THC (ng/g) | CBN (ng/g) | CBD (ng/g) | |
| Sample 1 | 16.2 | 4.6 | 0.0 | 6.0 | 0.0 | 0.5 | 0.0 | 0.0 | 0.0 | NA |
| Sample 2 | 537.0 | 8.5 | 4.0 | 33.8 | 53.2 | 8.7 | 1.2 | 0.3 | 0.0 | NA |
| Sample 3 | 122.0 | 3.0 | 0.0 | 7.3 | 0.0 | 4.1 | 0.2 | 0.2 | 0.2 | NA |
| Sample 4 | 89.9 | 4.1 | 3.7 | 9.9 | 11.4 | 3.3 | 0.1 | 0.3 | 0.2 | NA |
| Sample 5 | 79.4 | 3.9 | 3.3 | 7.6 | 0.0 | 2.7 | 0.3 | 0.2 | 0.1 | NA |
| Sample 6 | 84.5 | 2.4 | 0.0 | 3.6 | 0.0 | 0.8 | 0.3 | 0.0 | 0.0 | NA |
| Sample 7 | 157.0 | 2.3 | 0.0 | 7.3 | 0.0 | 2.0 | 0.0 | 0.0 | 0.2 | NA |
| Sample 8 | 15.2 | 1.4 | 2.2 | 3.2 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 9 | 14.7 | 1.4 | 0.0 | 4.7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 10 | 75.6 | 1.3 | 0.0 | 3.3 | 3.5 | 0.7 | 0.0 | 0.0 | 0.0 | NA |
| Sample 11 | 14.6 | 0.0 | 0.0 | 2.0 | 0.0 | 0.1 | 0.0 | 0.0 | 0.0 | NA |
| Sample 12 | 12.2 | 1.2 | 1.8 | 4.2 | 2.2 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 13 | 36.1 | 1.6 | 3.6 | 4.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 14 | 3.1 | 0.0 | 0.0 | 1.6 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 15 | 1.7 | 0.0 | 0.0 | 1.4 | 0.0 | 0.1 | 0.0 | 0.0 | 0.0 | NA |
| Sample 16 | 137.0 | 2.8 | 4.3 | 46.5 | 11.6 | 0.6 | 0.0 | 0.0 | 0.0 | NA |
| Sample 17 | 67.2 | 2.5 | 2.5 | 6.1 | 2.3 | 4.2 | 0.3 | 0.3 | 0.1 | NA |
| Sample 18 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 19 | 1.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 20 | 7.1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 21 | 1.4 | 0.0 | 0.0 | 0.7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 22 | 8.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 23 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 24 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 25 | 22.4 | 0.3 | 3.2 | 6.9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 26 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 27 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 28 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 29 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 30 | 101.0 | 2.0 | 0.0 | 19.0 | 3.0 | 3.0 | 0.4 | 0.0 | 0.1 | NA |
| Sample 31 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 32 | 6.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 33 | 41.0 | 0.0 | 0.0 | 4.0 | 0.0 | 0.4 | 0.0 | 0.0 | 0.0 | NA |
| Sample 34 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 35 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 36 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 37 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 38 | 60.0 | 0.0 | 1.4 | 6.8 | 2.3 | 2.5 | 0.1 | 0.0 | 0.1 | NA |
| Sample 39 | 17.0 | 0.0 | 0.0 | 0.3 | 0.0 | 0.7 | 0.0 | 0.0 | 0.0 | NA |
| Sample 40 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 41 | 18.0 | 0.0 | 0.0 | 9.0 | 0.0 | 0.2 | 0.2 | 0.0 | 0.0 | NA |
| Sample 42 | 20.0 | 2.0 | 0.0 | 9.0 | 0.0 | 1.9 | 0.2 | 0.0 | 0.1 | NA |
| Sample 43 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 44 | 2.1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
| Sample 45 | 370.0 | 2.0 | 3.0 | 20.0 | 5.0 | 3.9 | 0.1 | 0.0 | 0.0 | NA |
| Sample 46 | 38.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | NA |
Limitations of this study include (i) poor extraction efficiency for CBD in the umbilical cord samples, (ii) small numbers of paired specimens available, and (iii) a lack of information about the associated pregnancies/births. It is unknown, for example, whether any of the specimens tested in this study were associated with mothers known to use drugs, the term of the pregnancies, nor the outcomes of the newborns. It will be important that additional samples are analyzed to better understand the relationship between meconium and umbilical cord in newborns exposed to cannabis in utero. Although several studies have reported the agreement of drug screening results between cord and meconium, among certain common drug classes [31], [32], one published study reported results similar to that reported here [33]. These results accentuate the importance of establishing clinically significant cutoffs for these matrices. Relevant questions triggered by this inconsistency include: What sample type should be submitted for testing, and what is an appropriate cutoff to reduce false negatives while reducing the possibility of false positive results that could occur due to passive exposure? How would results change management decisions for the newborn and/or the mother? Is one specimen more likely to be positive based on the duration of the pregnancy or specific maternal drug use patterns? Would breast feeding be discouraged due to continued risk of exposure to cannabinoid analytes?
4. Conclusion
A LC-MS/MS method was developed and validated to detect and quantify THC, THCA, 11-OH-THC, CBN, and CBD in meconium and THC, THCA, 11-OH-THC, and CBN in umbilical cord. Concentrations and profiles of cannabinoid analytes varied considerably between umbilical cord and meconium samples. Concentrations of analytes in umbilical cord were much lower than in meconium, suggesting the need for different cutoffs to minimize false negative results. Analytes other than THCA were commonly observed, but the relevance of the profiles and concentrations of the individual analytes is not yet understood. These concentration and metabolite profiles could potentially help indicate time of exposure and characterize the potential impacts of in utero exposure to cannabis. In summary, the assay we describe here could help clinicians and researchers answer these and other questions by mapping clinical outcomes with analyte concentrations and abundance profiles in meconium and umbilical cord collected from cannabis-exposed newborns.
Funding
This work was supported by the ARUP Institute for Clinical and Experimental Pathology.
Conflict of interest
None of the authors has any conflicts of interest to disclose.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.clinms.2019.01.002.
Contributor Information
Triniti L. Jensen, Email: triniti.scroggin@aruplab.com.
Fang Wu, Email: fang.wu@saskhealthauthority.ca.
Gwendolyn A. McMillin, Email: gwen.mcmillin@aruplab.com.
Appendix A. Supplementary data
The following are the Supplementary data to this article:
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