Abstract
Opioid usage in the USA has increased over the past decade, with prescriptions increasing from 76 million in 1991 to 207 million in 2013. New regulations have curbed the number of prescriptions, leading to an increase in heroin use. Heroin-related overdoses have quadrupled between 2000 and 2015. The traditional urinary biomarkers for indicating heroin use are a combination of morphine and 6-acetyl morphine (6-AM). Morphine is detectable in urine for several days. 6-AM is detected in urine for 2–8 hours. Papaverine has been proposed as an alternative heroin biomarker. It has been reported to have a 1–2 day detection window. Papaverine metabolites have been reported to have up to a 3-day detection window. Presented is a method for the detection of papaverine and its metabolites, 6-desmethyl papaverine (6-DMP) and 4′, 6-didesmethyl papaverine (4,6-DDMP), in urine using a modified Waters® MCX™ microelution method. An ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS-MS), with a Waters’ BEH C18 column, and 20 mM ammonium formate water: 20 mM ammonium formate methanol mobile phase was employed. Calibration curves were linear from 0.1 to 50 ng/mL. No interferences were observed from the analysis of multicomponent therapeutic drug or drugs of abuse control materials; intra- and inter-run precision tests were acceptable. A total of 428 genuine urine specimens where heroin use was suspected were analyzed. These included 101 6-AM and 179 morphine only positive samples as well as 6 morphine-negative samples where papaverine and/or metabolites were detected. The determined concentrations in these samples for papaverine, 6-DMP and 4,6-DDMP ranged from 0.10 to 994, 0.10 to 462 and 0.12 to 218 ng/mL, respectively. The method was rugged and robust for the analysis of papaverine and metabolites, 6-DMP and 4,6-DDMP. The use papaverine and metabolites, 6-DMP and 4,6-DDMP has the potential to increase the detection window of heroin use.
Introduction
With the growing opioid epidemic, there is a renewed interest in the discovery and application of new biomarkers for illicit heroin use. Opioid usage in the USA has increased over the past decade with prescriptions increasing from 76 million in 1991 to 207 million in 2013. Recently, new regulations in many States have been enacted to curtail the number of opioid prescriptions physicians are writing. The resulting decrease in prescriptions has led to individuals increasingly turning to heroin as an opioid replacement, due to its increased availability and cheaper cost. The National Institutes of Health (NIH) reported a 5.9-fold increase in deaths involving heroin and non-methadone synthetic opioids from 2002 to 2015 (1). This is important because the 2016 National Health Interview Survey estimates that approximately 20% of the US population has chronic pain, and approximately 8% has high-impact chronic pain (2). The Institute of Medicine estimates that as of 2014, 100 million Americans were being treated for chronic pain and individuals being treated for pain have an 18–41% increase in risk for using illicit substances such as heroin. (3)
Monitoring or detecting heroin use in the pain management realm is therefore important to detect and potentially reduce heroin use before the individual becomes another statistic. Current heroin monitoring or detection involves analyzing for heroin metabolites, specifically 6-acetyl morphine (6-AM) and morphine. 6-AM is ideal in a post-mortem setting, but not in other settings, where detection indicates very recent use, due to its short half-life in the body. DAM (diamorphine, 3, 6-diacetyl morphine) is synthesized by acetylation of morphine that is extracted from opium produced by Papaver somniferum (poppy plant). DAM is unstable in aqueous solutions, and in people, it is rapidly hydrolyzed to 6-AM, and then to morphine by carboxylesterases in the liver, brain, plasma and erythrocytes (3). The half-life of DAM in the body is only 2–6 minutes (4). 6-AM has a reported detection window in urine of 2–8 hours. Therefore, morphine has been traditionally used, rather than DAM, to screen for heroin use, because morphine has a much longer detection window in urine, up to 3–4 days. However, morphine is not as specific for illegal heroin use, because morphine is also a metabolite of codeine (COD), and is an active pharmaceutical ingredient of many prescribed opiate formulations. Additionally, following poppy seed ingestion, morphine has been shown to be present in the urine for up to 2 days (5). Most testing for DAM use initially begins with a urine immunoassay screen for morphine. When morphine is detected via urine drug testing in the absence of other identifiable sources, such as prescribed opiates or ingestion of poppy seeds, clinicians presume heroin abuse by the patient (6).
In countries such as Germany and the UK that have heroin maintenance programs, individuals are prescribed and administered pharmaceutical grade DAM under controlled conditions (7). Therefore, 6-AM would be expected to be present in an individual’s urine. 6-AM would be an appropriate biomarker of DAM use but would not be an appropriate biomarker for illicit heroin use. Using 6-AM as a biomarker would allow the individuals to use pharmaceutical DAM as well as illicit heroin with no means of detecting the use of both. This is a critical distinction in these programs as patients are required to abstain from illicit heroin use as a condition of their continued treatment in the program. This has contributed to the need for alternative illicit heroin biomarkers.
The illicit production process of heroin rarely produces impurity-free DAM. Illicit heroin often contains other alkaloids extracted from the poppy plant, such as COD, papaverine (PAP), noscapine (NOS) and thebaine (THE) (8). These alkaloids and their respective metabolites have been evaluated as potential biomarkers for illicit heroin use. Since 2001, PAP has been the predominant alkaloid investigated. PAP has been found to constitute up to 20% of an illicit heroin sample (5). Even though less than 1% of PAP is excreted unchanged in urine (9), and PAP is only detectable 1–2 days post use, PAP metabolites have been detected in urine up to 7 days after illicit heroin use (7,10). The amount of PAP in opium can vary depending on the method used for extraction and heroin synthesis; and the geographical region where the poppy plant was grown. The predominant heroin synthesis method in Western Asia utilizes ammonia, which tends to increase the yield of PAP and NOS. The predominant heroin synthesis method in Southeast Asia utilizes lime, which tends to decrease the yield of PAP and NOS. The methods provide similar yields of morphine, COD, and THE. (8,10)
Two studies looked at opium alkaloids as potential biomarkers in inappropriately positive morphine urine specimens. PAP metabolites were consistently found and were not detected in the urine of morphine-negative patients. Neither PAP metabolites were present whether 6-AM was detectable or not (6,11). Pharmaceutical DAM preparations were also evaluated, as well as the patients that received them. Neither PAP nor its metabolites were detected in either (6,11). Other studies have noted that PAP was not in urine specimens after the individuals had consumed poppy seeds (5,6), but these studies were not analyzing for PAP metabolites. One study reported a sensitivity of 95.5% and specificity of 100% when comparing PAP metabolites to morphine as the gold standard for detection of heroin use (7), and another study reported that as many as five times more patients using illicit heroin were identified (11). A self-reporting study found some correlation between the time of heroin administration and the proportion of patients positive for PAP metabolites (7). A frequent objection to using PAP or its metabolites for heroin confirmation is their presence in poppy seed products. Opium alkaloids are present in both illegal heroin formulations and culinary products containing poppy seeds (13, 14). A study reported that immunoassay screens were positive for opiates up to 48 hours post ingestion of 60 g or one-half cup of poppy seeds and that two PAP metabolites were present in the subjects’ urine (5). In countries such as the USA, poppy seed use is generally relegated to low amounts in bagels or muffins even when added into the mix or placed on top as a garnish. In US populations where there is a potential or suspicion for misuse of opioid drugs such as in pain management patients, PAP metabolites can be useful if there is suspicion of illicit heroin use in the absence of any other explanation for the presence of morphine in an individual’s urine. In these scenarios, this testing would be useful as a reflex test. While PAP is not federally scheduled in the USA, the US Food and Drug Administration has no current approved uses, and in 1979, recommended its removal from the marketplace. However, PAP does have therapeutic uses in other countries, and some pharmaceutical preparations are known to be metabolized to PAP (14).
A potential limitation of testing for PAP and its metabolites is that although PAP has 15 potential demethylated metabolites, only 2 metabolites are currently commercially available as certified reference materials: 6-desmethyl papaverine (6-DMP; hydroxypapaverine desmethylpapaverine, 6-desmethylpapaverine), which is reported as the major urinary metabolite (15) and 4′,6-DDMP (dihydroxypapaverine, di-desmethylpapaverine, 4′,6-didesmethypapaverine) (6), which is a further demethylated metabolite (Figure 1). Isotopically labeled metabolites are also not commercially available for use as internal standards. Published methods for the detection of PAP involve gas chromatography mass spectrometry (5, 6, 7, 11, 12, 13) and are only qualitative in nature. Only one publication involved the use of liquid chromatography mass spectrometry (LCMS), and the method was only designed to detect PAP and NOS, not their metabolites (8). Recently, a method for synthesizing 13C labeled alkaloid metabolites for use as internal standards was published (16). The presented study is the only study that the authors are aware of that involves both the detection and quantitation of PAP and its 6-DMP and 4′,6-DDMP metabolites with detection using an ultra-performance liquid chromatography tandem mass spectrometry system (UPLC–MS-MS).
Figure 1.
Structure of PAP and its major metabolites.
Methods
Materials
The primary reference material for 4′,6-DDMP, 6-DMP, PAP and oxycodone-d6 (OC-d6) were obtained from Cerilliant Corporation (Round Rock, TX). Ammonium formate, ammonium hydroxide, glacial acetic acid, o-phosphoric acid and sodium acetate were ACS grade or better. Acetonitrile, methanol and water were LCMS grade or better. Oasis® MCX microelution plates were obtained from Waters Corporation (Milford, MA). Helix pomatia ß-glucuronidase was obtained from Sigma Aldrich (St. Louis, MO).
Instrumental analysis
The UPLC–MS-MS analysis was performed on a Waters AcQuity Xevo-TQ-S Micro UPLC–MS-MS system (Milford, MA) controlled by MassLynx 4.5 software. Chromatographic separation was performed on a Waters AcQuity UPLC® BEH C18 1.7 um, 2.1 × 50 mm column (Figure 2). The mobile phase consisted of 20 mM ammonium formate in water (A) and 20 mM ammonium formate in methanol (B) initially at 95:5 followed by a gradient to 60:40 by 1.5 minutes and 0:100 by 3.0 minutes then isocratic to 3.5 minutes and returned to 95:5. The column flow rate was 0.4 mL/min. Under these conditions, the retention times of O-d6, 4′,6-DDMP, 6-DMP and PAP were 1.3, 1.7, 2.0 and 2.3 minutes, respectively. The source and desolvation temperatures were 150°C and 600°C, respectively. Desolvation and cone gas flows were both 100 and 100 L/hr. The capillary voltage was 3.0 kV, and the ionization mode was positive electrospray. The acquisition mode used multiple reaction monitoring (MRM). The following transition ions (m/z) were monitored in MRM mode with their corresponding cone voltage (V) and collision energies (eV) in parentheses: OC-d6 (12): 322 > 218 (42), 247 (26) and 262 (24); 4′,6-DDMP (58): 312 > 128 (48), 158 (32) and 188 (24); and 6-DMP (40): 326 > 126 (205), 156 (32) and 188 (24); and PAP (36): 340 > 102 (66), 171 (32) and 202 (22). The total run time for the analytical method was 5.0 minutes.
Figure 2.
Total ion chromatogram, separation of OC-d6, 4,6-DDMP, 6-DMP, PAP and NOS.
Sample preparation
Sample preparation was by a modification of the Waters method for analyzing opiates in urine (15). Methanol was limited wherever possible except in the final elution step, due to the fact that methanol caused the PAP metabolites to elute from the microelution column before the final elution step. Four hundred picograms of OC-d6 (100 mcL, 4 mcg/mL in water) was added to 100 mcL of sample (calibration, QC, or specimen). Fifty microliters of ß-glucuronidase solution (200 mcL ß-glucuronidase (Helix pomatia) and 500 mcL 0.2 M acetate buffer, pH 5) was added. The solution was incubated at 58°C for at least 15 minutes. The solution was removed and allowed to cool to room temperature, then 100 mcL 4% o-phosphoric acid was added, and the solution was centrifuged at 10,000 g for 5 minutes at 10°C. Solid phase extraction was performed using a Waters Oasis® MCX microelution plate with a positive pressure manifold. Solutions were added in the following order and pushed through the plate at 7.5 psi after each solution, except for the drying step: 200 mcL methanol, 200 mcL water, supernatant of hydrolyzed sample, 200 mcL water twice and 200 mcL hexane. The plate was dried at 80 psi for 1 minute. The compounds were eluted and collected by adding 25 mcL of elution solvent (methanol:acetonitrile:ammonium hydroxide, 3:2:0.25) twice. One hundred microliters of water was added to the eluate, and the solution was transferred to an auto-sampler for analysis by UPLC–MS-MS. Ten microliters was injected for analysis.
Method validation
The evaluation of the urine QC material was conducted over five separate days (n = 15). Sample runs were analyzed as recommended for bioassay validation (9, 10) for linearity, lower limit of quantitation (LOQ), accuracy/bias, precision, carryover, selectivity, absolute recovery, matrix effect and stability. Validation sample runs contained calibrators in duplicate, drug-free control (negative control) with internal standard added and a double negative QC sample with no internal standard added, and the following QC samples containing 4′,6-DDMP, 6-DMP and PAP: limit of quantitation quality control (LOQC), target concentration of 0.1 ng/mL; low control (LQC), target concentration of 0.3 ng/mL; medium control (MQC), target concentration of 6 ng/mL; high control (HQC), target concentration of 40 ng/mL; and dilution control (DilQC; 1:4 dilution), target concentration 100 ng/mL.
Linearity, limit of quantitation and limit of detection
Linearity was verified from seven non-zero fortified drug-free urine matrices. These were prepared fresh in duplicate with concentrations of 0.1, 0.5, 1, 5, 10, 25 and 50 ng/mL each day for five separate days (n = 10). A linear regression of the ratio of the peak area of 6-DMP/OC-d6, 4′,6-DDMP/OC-d6 and PAP/OC-d6 versus concentration for each fortified sample of each separate set was used to construct the linear regressions to assess linearity, limit of quantitation and limit of detection (LOD). An acceptable criterion for the linearity, LOQ and LOD was a coefficient of determination (r2) of at least 0.98, and the calibrator concentrations within ±20% of their target value; and LOD and LOQ having a response at least 10 times greater than the signal-to-noise ratio of negative QC.
Accuracy/bias and precision
Accuracy/bias and precision were determined from the fortified QC samples (0.1, 0.3, 6, 40 and 100 ng/mL; 1:4 dilution). Intra-run precision was the run with the largest calculated intra-run % coefficient of variation (%CV) (n = 3) for each concentration over the five validation runs. Inter-run precision was calculated for each concentration over the five validation runs by using the combined QC values (n = 3) over the 5 days for a total of 15 replicates at each concentration. An acceptable bias would not exceed ±20% at each concentration with a precision having a coefficient of variation (%CV) of ≤ 15% except at the LOD of ≤ 20%.
Carryover
Sample carryover was evaluated in each of the five validation batches using two different procedures. First, immediately following the injection of the 50 ng/mL linearity material from each linearity set, either a drug-free control or a double negative control was injected. No carryover above the LOD was considered acceptable. Second, an injection of the HQC was immediately followed by injection of the LQC. This procedure was routinely applied each time the 50 ng/mL linearity material, HQC and LQC were analyzed with the determined concentration of the LQC at 85–115% of the mean. Carryover was also evaluated in the sample analysis batches in the following manner due to the high concentrations encountered in samples. This was accomplished by re-injecting the highest determined concentration samples and immediately following the injection of a drug-free control. No carryover above the LOD was considered acceptable.
Interferences
Elevated concentrations of common therapeutic and abused drugs as well as urine components were analyzed as part of the interference study (Table I). The materials were evaluated via a 1:1 mixing study with the 6 ng/mL and 40 ng/mL QC materials in the combined volume of 100 mcL. These samples were then analyzed in triplicate. Lack of observed interference was validated if the QC materials were within 25% of expected values.
Table I.
Common Therapeutic and Abused Drugs, and Urine Components Analyzed for Interferences and Specificity
| Bio-Rad Liquichek™ TDM Level 3 (Bio-Rad Laboratory, CA) | ||
|---|---|---|
| Acetaminophen | Amikacin | Amitriptyline |
| Caffeine | Carbamazepine | Chloramphenicol |
| Clonazepam | Cortisol | Cyclosporine |
| Desipramine | Diazepam | Digoxin |
| Disopyramide | Estriol | Ethosuximide |
| Flecainide | Gentamicin | Haloperidol |
| Ibuprofen | Imipramine | Lidocaine |
| Lithium | Methotrexate | N-acetyl Procainamide |
| Nortriptyline | Phenobarbital | Phenytoin |
| Primidone | Procainamide | Propranolol |
| Quinine | Salicylate | T3 |
| T4 | Theophylline | TSH |
| Valproic Acid | Vancomycin | |
| Bio-Rad Liquichek™ Urine Chemistry Control Level 2 (Bio-Rad Laboratory) | ||
| Amylase | Calcium | Uric Acid |
| Cortisol | Creatinine | Glucose |
| Magnesium | Microalbumin | Phosphorus |
| Potassium | Protein (total) | Sodium |
| Urea | Uric Acid | |
The following compounds were evaluated with the assay.
Selectivity
The selectivity of the assay was determined using 10 different urine specimens. Each specimen was analyzed with and without OC-d6. No peaks that co-eluted with the OC-d6, 6-DMP, 4′6-DDMP or PAP was considered acceptable. This ensured that compounds in the urines did not interfere with either the analytes or internal standard in the assay. Possible inter-urine matrix effects were determined by fortifying the 10 urine specimens with 0.3 ng/mL of 6-DMP, 4′6-DDMP and PAP. These samples were then analyzed in triplicate. Lack of observed interference was validated if the QC materials were within 25% of expected values.
Matrix Effect, Recovery and Process efficiency
The matrix effect, recovery and process efficiency of the assay for 6-DMP, 4′6-DDMP and PAP was determined at 0.25 and 4 ng/mL (n = 3) and for OC-d6, at 4 ng/mL (n = 6). Methanol was fortified with the compounds at the above concentrations. Drug-free urine was fortified with the compounds at the above concentrations. Drug-free urine was extracted following the procedure, and the eluate was fortified with the compounds at the above concentrations. The matrix effect was determined by comparing the mean absolute peak area of the eluate fortified samples to the mean absolute peak area of the drugs in methanol. The recovery was determined by comparing the mean absolute peak area of the eluate fortified samples to the mean absolute peak area of the extracted samples. The process efficiency of the assay was determined by comparing the mean absolute peak area of extracted samples to the mean absolute peak area of the drugs in methanol.
Storage and Freeze/Thaw Stability
The storage stability of 6-DMP, 4′6-DDMP and PAP was evaluated by storing the QC materials at refrigerator temperature (5°C) for 3 months and analyzing the QC materials on a biweekly basis. Bench-top stability was evaluated by storing the QC materials on the counter top at room temperature for 3 days to simulate if the materials were left unrefrigerated for up to 3 days (shipping or weekend). Freeze/thaw cycle stability was evaluated by freezing the LQC, and HQC materials, then allowing them to thaw unassisted, analysis in triplicate and then refreezing. This was repeated a total of three times. 6-DMP, 4′6-DDMP and PAP were considered stable if the concentrations of the LQC an HQC samples were within ±20% of their inter-run means.
Post-Preparative Stability
The post-preparative stability of OC-d6, 6-DMP, 4′6-DDMP and PAP was evaluated by leaving extracts of the LQC, and HQC sit in the UPLC–MS-MS’s auto-sampler. A batch (n = 3) of the extracted QCs was quantitated against a freshly prepared calibration. The extracted controls were then allowed to sit in the auto-sampler for 24 hours at 10°C. They were re-injected and quantitated using the initial calibration. The results of the initial analysis were compared with those of the re-injected samples. In the post-preparative study, OC-d6, 6-DMP, 4′6-DDMP and PAP were considered stable if the concentrations of the re-injected QC samples were within ±20% of their concentration determined by their initial injection.
Analysis of Pharmaceutical Morphine
The presence of 6-DMP, 4′,6-DDMP and PAP in pharmaceutical morphine preparations was determined. Fourteen lots of parenteral morphine preparations either commercially purchased or compounded by the VCU Health pharmacy, as well as one diacetyl morphine preparation obtained from the VCU Department of Pharmacology and Toxicology, were analyzed for their analyte concentrations.
Analysis of Poppy Seeds
The presence of 6-DMP, 4′,6-DDMP and PAP in poppy seeds was determined. Six lots of commercially available poppy seeds were analyzed for their analyte concentrations. These consisted of Penzeys Spices Whole Blue Holland, Frontier Certified Organic, NM Poppy Seeds, Sauers Whole, Spice Time Naturally Pure and Superstition Ranch Market. One gram of poppy seeds was incubated in 2 mL of deionized water at 58°C for 1 hour. The solution was centrifuged at 10,000 g for 5 minutes, and the supernatant was analyzed.
Analysis of Genuine Urine Specimens
The presence of 6-DMP, 4′,6-DDMP and PAP in genuine urine specimens was determined. A total of 428 de-identified previously opiate analyzed urine specimens from routine testing of pain management patients were sorted into three groups based on morphine and 6-AM content. The Heroin Positive group consisted of 101 urine specimens that containing both morphine and 6-AM. The morphine-positive group consisted of 179 urine specimens containing morphine but no 6-AM. The morphine-negative group consisted of 148 urine specimens containing no morphine or 6-AM, but contained other opioids and pharmaceuticals typically prescribed to pain management patients.
Results and discussion
The presented method demonstrates a procedure for the determination of PAP and its metabolites, 6-DMP and 4′,6-DDMP in urine. The method demonstrated acceptable accuracy and reproducibility. The method was linear from 0.1 to 50 ng/mL with the coefficients of determination (r2) in the 10 linearity sets yielding r2 = 0.9855–0.9999 for 6-DMP, and 4’,6-DDMP, r2 = 0.9833–0.9999 for PAP. The lower LOD and lower limit of quantitation (LOQ) were administratively set at 0.1 ng/mL. LOD and LOQ were within ±20% of the target value and had a response at least 10 times greater than the signal-to-noise ratio of negative QC. Based on initial work performed during the method development, if isotopically labeled internal standards were available, the LOD and LOQ for the assay would have been set at < 0.05 ng/mL. Accuracy as well as intra-day and inter-day precision of the assay were determined not to exceed CVs of ±20% over the dynamic range of the assay, except PAP (Table II). No analytical carryover was detected in any of the five analytical validation batches or sample analysis batches. No interferences were detected from common therapeutic and abused drugs or urine components. No interferences were detected from the 10 urine matrices. Fortification of the 10 urine matrices at 0.3 ng/mL ranged from 0.25 to 0.35 ng/mL (<1 SD). Process efficiency was determined at 0.25 ng/mL and 4.0 ng/mL ranged from 95% to 147% (Table III). QC materials were stable when stored at 5°C for up to 3 months. QC materials were stable when stored at up to 3 days at room temperature. QC materials were stable when processed through three freeze thaw cycles. QC material extracts stored on the UPLC–MS-MS were stable up to 24 hours after preparation. PAP and/or metabolites were not detected in pharmaceutical morphine and diacetylmorphine preparations. PAP and 6-DMP were present in some brands of poppy seeds, but at negligible levels (Table IV). Penzeys Spices Whole Blue Holland contained the highest amount of analyte with 292.6 ng PAP per gram of seed, which was approximately 7 times more than NM Poppy Seed (40.7 ng/g 6-DMP). This would equate to 1 mg PAP in approximately 3,425 grams or 7.5 pounds of poppy seeds. From the analysis of 428 pain management urine specimens (Table V), 89% of the Heroin Positive group contained at least 6-DMP, 4,6-DDMP and/or PAP, ranging from 0.1 to 993.9 ng/mL. In the morphine-positive group, 37% of the urine specimens contained at least 6-DMP, 4,6-DDMP and/or PAP, and 4% of the morphine-negative group contained at least 6-DMP, 4,6-DDMP and/or PAP. In the majority of these urines, 6-DMP was the most predominant compound detected. As with any testing scheme, there are limitations that can affect the overall effectiveness and efficiency of the scheme. The use of PAP and its metabolites as definitive biomarkers markers for illicit heroin use in the general population is not useful. This is due in part to the fact that there are no published methods the authors are aware of to date that have quantitated PAP metabolites in any matrix. Also, opiate alkaloid concentrations in poppy seed lots vary greatly around the world based on growing conditions, geographical location, plant variety and external contamination, but also on varying regulations as to their maximum allowable concentration in each country. However, their use as biomarkers to increase the detection window of potential illicit heroin use in defined populations is useful. In countries such as the USA, opiate alkaloid concentrations are very low as compared to the rest of the world. Their use by the population is generally relegated to low amounts in bagels or muffins. Therefore, in populations where there is a potential or suspicion for misuse of opioid drugs such as in pain management patients and/or in historically heroin prevalent cities, they can be useful if there is suspicion of illicit heroin use in the absence of any other explanation for the presence of morphine in an individual’s urine. In these scenarios, this testing would be useful as a reflex test. As with all testing, understanding the necessity and usefulness is important to the interpretation of results to ensure correct understanding of not only the validity and accuracy of the testing method, but also application of the results.
Table II.
Assay Precision Inter- and Intra-run Bias and %CVs
| Intra-day precision (n = 3) | |||
|---|---|---|---|
| Control: x̅ (%CV) | Control: x̅ (%CV) | 4′,6-DDMP | PAP |
| LOQ (0.1 ng/mL) | 0.11 (19) | 0.14 (3) | 0.12 (16) |
| LQC (0.3 ng/mL) | 0.26 (11) | 0.28 (7) | 0.38 (14) |
| MQC (6 ng/mL) | 6.6 (14) | 5.9 (10) | 6.5 (13) |
| HQC (40 ng/mL) | 43.9 (10) | 40.4 (3) | 41.6 (11) |
| DilQC (100 ng/mL) | 107.8 (9) | 101.5 (2) | 85.8 (12) |
| Inter-day precision (n = 15) | |||
| Control: x̅ (%CV) | 6-DMP | 4′,6-DDMP | PAP |
| LOQ (0.1 ng/mL) | 0.10 (17) | 0.11 (20) | 0.12 (19) |
| LQC (0.3 ng/mL) | 0.27 (14) | 0.28 (13) | 0.30(15) |
| MQC (6 ng/mL) | 5.7 (12) | 5.7 (14) | 6.2 (8) |
| HQC (40 ng/mL) | 35.3 (12) | 36.4 (10) | 37.2 (11) |
| DilQC (100 ng/mL) | 85.6 (15) | 88.9 (13) | 97.9 (11) |
Table III.
Matrix Effect, Recovery and Process Efficiency with the %CV in Parentheses (n = 6), Determined at Two Concentrations
| 6-DMP | 4′,6-DDMP | PAP | OC-d6 | |
|---|---|---|---|---|
| Matrix effect (%) | ||||
| 0.25 ng/mL | −1 (9) | −2 (8) | 10 (9) | |
| 4.0 ng/mL | 0 (8) | −2 (6) | 7 (8) | 37 (7) |
| Recovery (%) | ||||
| 0.25 ng/mL | 143 (9) | 132 (8) | 140 (8) | |
| 4.0 ng/mL | 108 (9) | 103 (6) | 110 (10) | 108 (8) |
| Process efficiency (%) | ||||
| 0.25 ng/mL | 70 (9) | 74 (8) | 78 (8) | |
| 4.0 ng/mL | 93 (8) | 96 (5) | 98 (9) | 147 (7) |
Table IV.
Prevalence of PAP and Metabolites in Poppy Seeds and Pharmaceuticals
| Brand | 6-DMP (ng/g) | 4′,6-DDMP (ng/g) | PAP (ng/g) |
|---|---|---|---|
| Frontiers Certified Organic | 2.9 | ND | 10.9 |
| NM Poppy Seed | 40.7 | ND | ND |
| Penzeys Spices—Holland | 1.8 | ND | 292.6 |
| Sauers—Whole | 3.0 | ND | ND |
| Spice Time—Naturally Pure | ND | ND | ND |
| Superstition Ranch Market | ND | ND | ND |
| (mg/g) | (mg/g) | ||
| Frontiers | 0.0000029 | 0.000011 | |
| NM Poppy Seed | 0.0000407 | ND | |
| Penzeys | 0.0000018 | 0.000292 | |
| Sauers—Whole | 0.0000030 | ||
| Pharmaceuticals | |||
| Morphine (14 lots) | ND | ND | ND |
| Diacetylmorphine | ND | ND | ND |
Table V.
Prevalence of PAP and Metabolites in Urine Samples (ng/mL)
| Group | Pos (n) | Analyte | Mean | Median | Range (ng/mL) |
|---|---|---|---|---|---|
| 80 | 6-DMP | 97.3 | 38.4 | 0.2–762.9 | |
| Heroin pos | 64 | 4′,6-DDMP | 16.8 | 13.1 | 0.6–218.0 |
| (T/Pos = 101/90) | 45 | PAP | 22.2 | 1.3 | 0.1–993.9 |
| Morphine pos | 51 | 6-DMP | 6.8 | 9.2 | 0.1–462.1 |
| (Heroin Neg) | 35 | 4′,6-DDMP | 1.7 | 4.2 | 0.1–104.8 |
| (T/Pos = 179/67) | 20 | PAP | 0.1 | 0.5 | 0.1–7.3 |
| 5 | 6-DMP | 8.3 | 3.9 | 0.2–20.7 | |
| Morphine Neg | 3 | 4′,6-DDMP | 3.2 | 4.2 | 0.8–4.6 |
| (T/Pos = 148/6) | 3 | PAP | 0.5 | 0.2 | 0.1–1.1 |
T/Pos = Total # samples in group/total # PAP, 6-DMP and/or 4′-6-DMP positive samples.
Conclusion
The presented method for the determination of 6-DMP, 4,6-DDMP and PAP in urine is rugged and robust. This method was used to analyze pharmaceutical, poppy seeds and genuine pain management urine specimens for the presence of 6-DMP, 4,6-DDMP and PAP, to determine if they are plausible biomarkers for the identification of illicit heroin use. No common therapeutic or abused drug or urine constituent interfered with the analysis. QC materials were stable under all tested storage conditions. No analytes were detected in the parenteral pharmaceutical preparations tested. 6-DMP and PAP were detected in poppy seeds tested; however their presence in the urine specimens that were tested, being attributed to poppy seed consumption, is highly unlikely, due to the low presence of opiate alkaloids as compared to other countries in the world. 6-DMP, 4,6-DDMP and/or PAP were detected in 89% of the 6-AM positive urine specimens, 37% of the morphine-positive urine specimens and only present in 4% of the morphine and 6-AM negative urine specimens. This along with the fact that PAP is not routinely used medically in the US indicates that when used in conjunction with 6-AM and morphine, 6-DMP, 4,6-DDMP and PAP are plausible biomarkers for illicit heroin use in the USA.
Funding
This project was funded in part by the National Institute of Justice, Research and Development in Forensic Science for Criminal Justice Purposes, 2016-DN-BX-0148 and 2017-R2-CX-0029, the National Institute of Health (P30DA03393), and the Virginia Commonwealth University Undergraduate Research Opportunities Program (UROP).
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