Stability of 21 Cocaine, Opioid and Benzodiazepine Drug Analytes in Spiked Meconium at Three Temperatures

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1 Journal of Analytical Toxicology, 2017;41: doi: /jat/bkw113 Advance Access Publication Date: 26 November 2016 Article Article Stability of 21 Cocaine, Opioid and Benzodiazepine Drug Analytes in Spiked Meconium at Three Temperatures Fang Wu 1, Stephanie J. Marin 2, and Gwendolyn A. McMillin 1,2, * 1 Department of Pathology, University of Utah, Salt Lake City, UT 84132, USA, and 2 ARUP Laboratories, 500 Chipeta Way, MS-115, Salt Lake City, UT 84108, USA *Author to whom correspondence should be addressed. gwen.mcmillin@aruplab.com Abstract In this study, the stability of 21 cocaine, opioid and benzodiazepine analytes in spiked meconium was investigated at three storage temperatures: 4 C, room temperature (RT), and 37 C (body temperature). The drugs/metabolites included were hydrocodone, hydromorphone, codeine, morphine, 6-acetylmorphine (6-AM), oxycodone, oxymorphone, cocaine, cocaethylene, benzoylecgonine, m-hydroxybenzoylecgonine, diazepam, oxazepam, temazepam, nordiazepam, chlordiazepoxide, lorazepam, alprazolam, alpha-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, midazolam, alpha-hydroxymidazolam and zolpidem. Drug testing was performed using mass spectrometry methods that were validated for clinical use. After 2 weeks of storage, a substantial loss was observed in the concentrations of 7-aminoclonazepam (48.4% at 4 C and 71.5% at RT), and chlordiazepoxide (59.5% at RT). A slight decrease was observed in the concentrations of alprazolam (20.9% at 4 C), clonazepam (24.5% at 4 C), chlordiazepoxide (23.5% at 4 C), midazolam (20.8% at 4 C), nordiazepam (22.8% at RT), and alpha-hydroxyalprazolam (20.7% at 4 C). At 37 C, the concentrations of chlordiazepoxide, 7-aminoclonazepam, lorazepam, oxazepam, nordiazepam and temazepam decreased by 81.4%, 86.8%, 56.5%, 59.9%, 45.4% and 31.7%, respectively, after 2 weeks. 6-AM was observed to be unstable regardless of storage temperatures. For morphine, a 33.3% increase at 4 C and a 23.4% increase at RT were observed after 2 weeks, respectively, possibly due to 6-AM degradation, while no changes 20% were observed at 37 C. All other analytes were stable up to 2 weeks at all three storage temperatures (concentration changes <20%). The stability of select drug analytes in authentic clinical meconium specimens was consistent with that observed in spiked meconium. In conclusion, some drugs in meconium may not be stable for long periods of time. Sample storage conditions are an important consideration in the context of detection windows and interpreting drug-testing results in meconium. To the best of our knowledge, this is the first stability study of cocaine, opioids and benzodiazepines in meconium concerning the effects of storage temperatures. Introduction Newborns exposed to drugs in utero may exhibit symptoms of preterm delivery, low birth weight, neonatal withdrawal syndromes, as well as other short- and long-term health problems (1 4). Drug testing is a useful tool to identify drug exposure and help clinicians develop a treatment plan for these babies, since it is inadequate to rely on maternal self-reporting alone (5). Meconium has become a well-established specimen to assess in utero drug exposure due to its presumed wide detection window and the ease of collection, although other biological specimen types, such as neonate urine, The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com 196

2 Drug Stability in Meconium 197 hair and umbilical cord tissue, have been used to detect fetal drug exposure (6, 7). Formation of meconium begins between the 12th and 13th gestational week until birth (8), and would theoretically reflect drug use during the second and third trimesters (9). A large number of studies have been published on analyzing meconium to identify maternal drug use, such as amphetamines (10), opioids (11, 12), cocaine (11) and cannabis metabolites (13, 14), finding that actual detection window varies. Detection window for meconium may be influenced by drug stability in utero, as well as drug stability during storage and transport. Indeed, the stability of drugs is a major concern during the evaluation of toxicological results (15, 16). The concentration changes of analytes during transportation to the laboratory under inappropriate transportation temperatures could overestimate or underestimate the concentrations of drugs in specimens and ultimately affect qualitative detection of drug analytes and final interpretation of results. It is not uncommon for a few days to pass between the time of sample collection and laboratory analysis. The knowledge of drug stability could aid the result interpretation, as well as define the optimal sample storage conditions. By far, most published data on the stability of drugs mainly focused on the effects of storage temperatures on the biological sample types plasma/serum, urine, hair and oral fluids (17 20). Peters et al. has provided a comprehensive overview of the in vitro stability of drugs in blood, plasma and serum samples (21). Literature search revealed little published data on the stability of drugs and/or drug metabolites in meconium. We only identified one study that presented a 25% decrease in cocaine and cannabinoid concentration after meconium was allowed to stand at room temperature (RT) for 24 hours (22). The objective of this study is to investigate the stability of 21 cocaine, opioid and benzodiazepine analytes in meconium at three storage temperatures (4 C, RT and 37 C), after 3, 7 and 14 days of storage. Three fully validated mass spectrometry methods were used to determine the concentration of each analyte at each storage temperature (11, 12, 23). The drugs/metabolites included in the opioid assay (liquid chromatography tandem mass spectrometry, LC MS/MS) were hydrocodone, hydromorphone, codeine, morphine, 6-acetylmorphine (6-AM), oxycodone and oxymorphone; cocaine, cocaethylene, benzoylecgonine and m-hydroxybenzoylecgonine were included in the cocaine assay (gas chromatography mass spectrometry, GC-MS); diazepam, oxazepam, temazepam, nordiazepam, chlordiazepoxide, lorazepam, alprazolam, alphahydroxyalprazolam, clonazepam, 7-aminoclonazepam, midazolam, alpha-hydroxymidazolam and zolpidem were included in the benzodiazepine assay (LC MS/MS), which included pre-analytical hydrolysis. Of note, the results from storage at 37 C (body temperature) could negatively impact the drug detection window of meconium in utero. Methods and materials Chemicals and reagents All solvents used were reagent grade or better and were purchased from VWR International (West Chester, PA, USA) or Thermo Fisher Scientific (Waltham, MA, USA), including 2-propanol, methanol and acetonitrile. The following reagents were purchased from Sigma- Aldrich (St. Louis, MO, USA): formic acid, ammonium formate, ammonium hydroxide, sodium chloride, sodium bicarbonate, sodium phosphate, methylene chloride, ethyl acetate and acetic acid. Drug standards for the target analytes were purchased from Cerilliant (Round Rock, TX, USA). The internal standards (ISs) were also purchased from Cerilliant: cocaine-d3, cocaethylene-d8, benzoylecgonined8, alprazolam-d5, chlordiazepoxide-d5, clonazepam-d4, diazepam-d5, alpha-hydroxyalprazolam-d5, lorazepam-d4, midazolam-d4, nordiazepam-d5, oxazepam-d5, temazepam-d5, zolpidem-d6, codeine-d6, morphine-d6, hydromorphone-d3, oxycodone-d6, oxymorphone-d3 and 6-AM-d6. Beta-glucuronidase was purchased from Campbell Science (Rockford, IL, USA). N-Methyl- N-trimethylsilyl-trifluoroacetamide was purchased from United Chemical Technologies, Inc. (Bristol, PA, USA). Type I water was generated using a Barnstead Nanopure Infinity ultrapure water system (Thermo Fisher Scientific). Spiked sample preparation Drug-free meconium was created by pooling more than 30 residual patient samples that had been previously tested negative for the drug analytes included in this study. Samples were deidentified according to institutional protocols. The three mass spectrometry methods applied here were validated for clinical use as described previously (11, 12, 23). Based on the assays, either 0.25 or 0.5 g of drug-free meconium was weighed into individual tubes (n = 150). Drug standard solutions were then spiked into individual tubes to produce an equal concentration of each analyte that is detected in the three assays: 200 ng/g (cocaine), 200 ng/g (opioids) and 50 ng/g (benzodiazepines). The target concentrations were chosen because they approximate median concentrations observed in authentic clinical specimens sent to our laboratory for routine testing (24). Furthermore, the target concentrations fall comfortably within the analytical measurement ranges of the assays, to assure that changes in concentrations could be detected. Next, each spiked sample was rigorously vortexed and mixed until uniform, consistent with routine protocols for preparation of matrix-matched calibrators and quality-control materials. The samples were then stored refrigerated at 4 C (2 8 C), ambient or RT (23 25 C) and at body temperature 37 C (36 38 C). Environmental controls assure consistency and documentation of temperatures, as per institutional protocols. For cocaine and benzodiazepines, 60 aliquots of spiked meconium were prepared (30 per drug class). At the 0 day time point, assays were performed in triplicate for each drug class at RT (without considering the effects of storage temperatures; n = 3 2 = 6). For the 3, 7 and 14 days time points, assays were performed in triplicate for each of the three storage temperatures (n = = 54). For opioids, 90 aliquots of spiked meconium were prepared and analyzed in three separate runs (30 per run). Due to a run failure at 14 days, the opioid data from the failed run (n = 9) were unavailable. The workflow is summarized in Supplementary Figure 1. At the designated storage time at each temperature, samples were stored frozen ( 20 C) until tested. The samples were then analyzed as previously described (11, 12, 23). Briefly, samples were thawed and deuterated analogs of the compounds of interest were added to the meconium as ISs. The samples were then homogenized in methanol using mm stainless steel beads (Next Advance, INC., Averill Park, NY, USA) and a Bullet Blender homogenizer (Next Advance, INC.). Solid phase extraction was performed using Trace-J SPE columns from SPEWare (Baldwin Park, CA, USA), as described previously (11). The eluents were dried down and reconstituted in appropriate solvent. For benzodiazepines, the assay includes preanalytical enzymatic hydrolysis (23). Cocaine and metabolites were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide after solid phase extraction. The derivatized solution was then subjected

3 198 Wu et al. to GC-MS analysis with three calibrators containing 20, 50 and 200 ng/g of each analyte. One positive and one negative control were prepared in the assay. In the opioid and benzodiazepine assays, four calibrators were used, containing 20, 50, 200 and 1000 ng/g of each analyte. One negative and two positive controls (i.e., low positive and high positive) were included in the opioid and benzodiazepine assays. Calibrators, positive and negative controls were prepared and analyzed under the same conditions with each sample set. The lower limit of quantification (LOQ) was 20 ng/g for all analytes except alprazolam and alpha-hydroxyalprazolam, clonazepam and 7-aminoclonazepam, and diazepam, for which the LOQ was 5 ng/g. Of note, the deuterated IS for m-hydroxybenzoylecgonine was not commercially available, so this compound was referenced to the benzoylecgonine IS. All other compounds were referenced to a dedicated deuterated IS. Authentic clinical specimen preparation The stability of seven drug analytes (i.e., alprazolam, lorazepam, hydrocodone, oxycodone, oxymorphone and benzoylecgonine) was investigated using pooled residual patient specimens that tested positive in our laboratory. Five meconium pools were generated: (i) meconium pool #1: by pooling three patient specimens that tested positive for lorazepam and alprazolam; (ii) meconium pool #2: by pooling five patient specimens that tested positive for morphine only; (iii) meconium pool #3: by pooling three patients specimens that tested positive for oxycodone and oxymorphone; (iv) meconium pool # 4: by pooling two patient specimens that tested positive for hydrocodone only; (v) meconium pool #5: by pooling two patient specimens that tested positive for benzoylecgonine only. Next, individual meconium pool was homogenized as described for the spiked samples. Each meconium pool was then aliquoted and stored refrigerated at 4 C (2 8 C), ambient or RT (23 25 C) and at body temperature 37 C (36 38 C). Due to the limited amount of residual specimens, only two time points (0 and 14 days) were investigated. At the 0 day time point, assays were performed in triplicate at RT (without considering the effects of storage temperatures; n = 3 5 = 15). At the 14 days time point, assays were performed in triplicate for each of the three storage temperatures (n = = 45). At the designated storage time at each temperature, samples were stored frozen ( 20 C) until tested. The samples were then analyzed in the same manner as the spiked meconium as described above. Mass spectrometry analysis Cocaine and metabolites were analyzed by an Agilent Technologies 6890 N/5973 electron impact gas GC-MS (Santa Clara, CA, USA) equipped with a DB-5 ms (0.25 i.d. 15 m length) capillary column (VWR International) and an autosampler (Agilent). Agilent Technologies Chemstation software was used to control the instrument. The GC-MS was operated in the selected ion monitoring (SIM) mode. Benzodiazepines and opioids were analyzed by AB SCIEX Triple Quad TM 5500 mass spectrometer (Framingham, MA, USA) interfaced with CTC PAL HTC-xt-DLW autosampler (Lake Elmo, MN, USA) and Agilent 1260 infinity series binary pump, degasser and column oven as described previously (11, 12, 23). Separation was performed on an Agilent Zorbax Eclipse Plus C18 ( mm, 1.8 µm particle size) RRHD column (Agilent). The instrument was operated in positive electrospray ionization mode. Data were collected in scheduled multiple reaction monitoring mode. AB SCIEX Analyst software (version 1.5.2) was used for instrument control and data analysis. Two transition ions were used to monitor each drug analyte. Quantitation was performed using Ascent Indigo software (Indigo Bioautomation, Indianapolis, IN, USA). Data analysis The concentration of each analyte was determined based on the calibration curve generated with each batch of samples. The data were imported into Excel to perform analysis. The concentration at each time point was compared to the initial concentration, determined with the 0 day time point. Concentrations were considered equivalent if within 20% of the initial concentration, reflecting absolute differences when the imprecision of routine clinical meconium testing is accounted for (coefficient of variation of 20 30%). In our laboratory, meconium testing is associated with higher imprecision than other matrices (e.g., urine). The highest imprecision was observed for m-hydroxybenzoylecgonine, due in large part to the lack of an isotopically labeled analog to reference as an IS. Results Stability of benzodiazepines in spiked meconium Alprazolam, alpha-hydroxyalprazolam, midazolam and alphahydroxymidazolam: All 4 analytes showed good stability, achieving concentrations within approximately 20% of the original, at all three storage conditions and all time points (Figure 1). Chlordiazepoxide: The concentrations of chlordiazepoxide decreased at all storage conditions. Under storage temperatures of RT and 37 C, significant losses were found after 2 weeks of storage with 39.5% and 81.4% loss, respectively. Chlordiazepoxide was relatively stable at 4 C in that the mean concentration decreased by 23.5% after 2 weeks storage. Clonazepam and 7-aminoclonazepam: At all storage temperatures, clonazepam was stable over a 2-week storage period. 7- Aminoclonazepam, a major metabolite of clonazepam, was found to be unstable at all storage temperatures. At 4 C, the mean concentration of 7-aminoclonazepam decreased by 25.5%, 39.8% and 48.4% at 3, 7 and 14 days, respectively; at RT, the mean concentration changes were 48.6%, 41.1% and 71.5% at 3, 7 and 14 days, respectively. A dramatic change was observed at 37 C in that the mean concentration of 7-aminoclonazepam was found to decrease by 86.8% at 14 days. Diazepam, temazepam, nordiazepam and oxazepam: At 4 C and RT, a slight decrease (<25%) was observed after 2 weeks of storage. However, at 37 C, the mean concentrations were found to decrease by 59.9% (oxazepam), 30.7% (temazepam) and 45.4% (nordiazepam) after 2 weeks of storage, respectively. For diazepam, only a minor concentration loss (<25%) was observed at 37 C over a 2-week storage period. Lorazepam: At 37 C, lorazepam decreased by 13.9%, 27.6% and 56.5% at 3, 7 and 14 days, respectively, while at 4 C and RT, lorazepam showed good stability (<20% change) over a 2 week storage period. Zolpidem: Stable up to 2 weeks regardless of storage temperatures. Stability of opioids in spiked meconium 6-AM was the most unstable analyte with loss of ~50% after 2 weeks at any storage temperature. For morphine, the mean concentration was stable at 37 C while an approximate 30% increase in the mean concentrations was found at 4 C and RT after 2 weeks of storage. All other analytes (i.e., codeine, hydrocodone,

4 Drug Stability in Meconium 199 hydromorphone, oxycodone and oxymorphone) were stable up to 2 weeks of storage at all conditions (Figure 2). Stability of cocaine and metabolites in spiked meconium All drug analytes were stable at all temperature conditions and time points (Figure 3). Stability of lorazepam, alprazolam, morphine, hydrocodone, oxycodone, oxymorphone and benzoylecgonine in authentic clinical specimens Stability of the seven drug analytes investigated (i.e., lorazepam, alprazolam, morphine, hydrocodone, oxycodone, oxymorphone and benoylecgonine) in authentic meconium was consistent with the findings from the spiked meconium study. The results are illustrated in Supplementary Figure 2. Alprazolam, hydrocodone and Figure 1. Time-course changes in the concentrations of (A) diazepam, (B) oxazepam, (C) temazepam, (D) nordiazepam, (E) chlordiazepoxide, (F) lorazepam, (G) alprazolam, (H) alpha-hydroxyalprazolam, (I) clonazepam, (J) 7-aminoclonazepam, (K) midazolam, (L) alpha-hydroxymidazolam and (M) zolpidem under three storage conditions ( ) 4 C, ( ) RT and ( ) 37 C, presented as mean percentages of day 0 recoveries ± standard deviation. Thirty aliquots of spiked meconium were prepared. At the 0 day time point, assays were performed in triplicate at RT (without considering the effects of storage temperatures; n = 3). For the 3, 7 and 14 days, assays were performed in triplicate at each of the three storage temperatures (n = = 27).

5 200 Wu et al. Figure 1. Continued benzoylecgonine showed good stability (<20% change) over 2 weeks of storage at all conditions; for lorazepam, after 14 days of storage, the mean concentration decreased by 81.6% at 37 C while lorazepam showed good stability (<20% change) at 4 C and RT. For morphine, the mean concentration slightly changed at RT and 37 C after 14 days of storage (<30% change). At 4 C, morphine was stable with the mean concentration change <10% at the 14 days time point. The mean concentrations of oxycodone and oxymorphone were found to slightly increase (15 36%) at all three temperatures at the 14 days time point. Discussion Meconium has become a well-established specimen to assess prenatal drug exposure in the past 20 years (7, 25). Since it accumulates from the second trimester until birth, meconium analysis is thought to detect maternal drug use during the second and third trimesters. However, the in utero drug detection window is not well studied. Both the in vivo and the in vitro stability of drugs in meconium could influence the detection window of specific drug analytes in meconium. Literature searching identified little information on the stability of drugs/drug metabolites in meconium in vitro and in vivo (22). In this study, the stability of 21 cocaine, opioid and benzodiazepine analytes in meconium was investigated at three storage temperatures: 4 C, RT and 37 C (body temperature). The results from the storage conditions at 4 C and RT represented the in vitro stability that is relevant to storage and handling of specimens from the time of collection through analysis; the results from the storage condition at 37 C represented the detection window of meconium in vivo. To our knowledge, this is the first stability study of cocaine, opioids and benzodiazepines in meconium concerning the effects of storage temperatures. The stability of benzodiazepines under different storage conditions has been well studied in urine (26), plasma (27, 28) and whole blood (27 30), but not in meconium. In urine specimens (26), after 30 days of storage at 4 C, alprazolam, nordiazepam and oxazepam showed good stability with the recovery of 95%, 90 98% and 97% while the recovery of alpha-hydroxyalprazolam and temazepam was relatively lower ranging from 43% to 85%. In whole blood specimens, the concentrations of clonazepam and midazolam were found to decrease to 60% after a year of storage at RT and at 4 C (27). In agreement with the previous studies (26), our data showed good

6 Drug Stability in Meconium 201 Figure 2. Time-course changes in the concentrations of (A) hydrocodone, (B) hydromorphone, (C) codeine, (D) morphine, (E) 6-AM, (F) oxycodone and (G) oxymorphone under three storage conditions ( ) 4 C, ( ) RT and ( ) 37 C, presented as mean percentages of day 0 recoveries ± standard deviation. Ninety aliquots of spiked meconium were prepared and analyzed in three separate runs (30 per run). At the 0 day time point, assays were performed in triplicate at RT (without considering the effects of storage temperatures; n = 3 3 = 9). For the 3, 7 and 14 days, assays were performed in triplicate at each of the three storage temperatures (n = = 81). Due to a run failure at 14 days, the opioid data from the failed run (n = 3 3 = 9) were unavailable for data analysis. stability of alprazolam, nordiazepam and oxazepam with a minor loss (<25%) in meconium at 4 C and RT over 2 weeks of storage. However, inconsistent with the previous studies (26, 27), our data showed good stability of temazepam, clonazepam, midazolam and alpha-hydroxyalprazolam with mean concentration change <20% over 2 weeks of storage at 4 C and at RT. We believe that the discrepancies between the results of our study and the previous studies (26, 27) might be attributed to the different storage periods and the different biological matrices. Recently, Atanasov et al. (30) studied the effects of storage conditions on the stability of diazepam. The

7 202 Wu et al. Figure 3. Time-course changes in the concentrations of (A) cocaine, (B) cocaethylene, (C) benzoylecgonine and (D) m-hydroxybenzoylecgonine under three storage conditions ( ) 4 C, ( ) RT and ( ) 37 C, presented as percentages of day 0 recoveries ± standard deviation. Thirty aliquots of spiked meconium were prepared. At the 0 day time point, assays were performed in triplicate at RT (without considering the effects of storage temperatures; n = 3). For the 3, 7 and 14 days, assays were performed in triplicate at each of the three storage temperatures (n = = 27). data showed a 30% decrease in the concentration of diazepam in blood samples after 4 weeks of storage at 4 C (30). In agreement with the results published by Atanasov et al. (30), our data showed a 20 30% decrease in the concentration of diazepam in meconium at all three temperatures. Although the degradation products of many analytes have been well studied in pharmaceutical preparations, little information was found regarding the prevalence and mechanism of spontaneous degradation or degradation by enzymatic metabolism in meconium. In the case of chlordiazepoxide, the concentration of this particular drug decreased by ~80% after 2 weeks of storage at 37 C. However, the concentration of commonly expected metabolites of chlordiazepoxide (i.e., nordiazepam and oxazepam) did not increase. The findings suggest that detection of maternal chlordiazepoxide use during pregnancy will require targeting alternative degradation products in meconium. In contrast, we found that the concentration of midazolam decreased while the concentration of alpha-hydroxymidazolam, the major metabolite of midazolam, increased after 2 weeks of storage at all three temperatures. The data might suggest that enzymatic metabolism of midazolam may occur in meconium. Stability of drug analytes is commonly shown to be temperaturedependent, wherein frozen storage provides the highest degree of stability, and warmer storage conditions may be associated with poor stability. This trend was observed with many of the drug analytes included in this study, as described above for benzodiazepines. Other drug analytes were stable regardless of temperatures. Among the opioid analytes, hydrocodone, hydromorphone, oxycodone, oxymorphone and codeine were stable up to 2 weeks of storage regardless of storage temperatures. Our results are consistent with the previous stability study on these four analytes in plasma samples published by Musshoff et al. (31). In our study, 6-AM was the most unstable analyte in that the mean concentrations dramatically decreased regardless of storage temperatures, which agreed with the results published previously (26). The stability of morphine in plasma has been studied elsewhere (31, 32). No indication of instability of morphine was observed in these studies (31, 32). However, our data showed a substantial increase at 4 C and RT while, at 37 C, morphine in meconium was found to be stable over 2 weeks of storage. The interesting stability pattern of morphine might reflect a combination of hydrolysis of 6-AM to morphine, and degradation of morphine. Our hypothesis is that morphine is degraded at a slower rate than 6-AM, and that the degradation of morphine is temperature-dependent. As such, morphine was generated from 6-AM at 37 C, as with other storage temperatures, but at 37 C morphine degraded to a concentration that approximates 100% of the expected value. Meanwhile, morphine generated from 6-AM at 4 C and RT was not degraded and accumulated such that the concentration of morphine increased after 2 weeks of storage. Additional studies would be required to evaluate that hypothesis including the use of authentic patient samples versus prepared (spiked) samples. Nonetheless, the instability of 6-AM suggests that maternal heroin use may not be specifically detected in meconium that is collected more than 2 weeks after last use. Previous studies using authentic samples have suggested that detection of heroin use in meconium relies on detection of metabolites such as morphine (24). The stability of cocaine and its metabolites has been studied in a number of specimen types, including whole blood (33, 34), plasma (33), urine (35, 36), saliva (37) and tissues (38). Javaid et al. (36) reported that there was a 80% degradation of cocaine at 37 C and only a 14% decrease at 4 C in urine samples. In another study, cocaine was found to be unstable in blood, especially when stored at RT (34). Our findings were contradictory with the previous studies

8 Drug Stability in Meconium 203 in that cocaine, cocaethylene and benzoylecgonine were found to be stable up to 2 weeks at all three storage temperatures with the mean concentration changes <10%. We identified one study that reported a 25% decrease in the concentration of cocaine in meconium after 24 hours of storage at RT (22). Again, the discrepancies could be attributed to the difference specimen types and the different storage periods between our study and these previous studies (34, 36). For m-hydroxybenzoylecgonine, we found a 20 30% decrease at day 3 at 4 C, RT and 37 C while an increase (10 30%) was observed at all three temperatures at the 14 days time point. This observation may represent analytical artifact because this particular analyte is associated with larger imprecision than other cocaine analytes in this method, most likely due to the lack of a dedicated IS. Imprecision in the data also reflects the relatively small number of samples tested in this study. To our knowledge, this is the first stability study in meconium concerning the effects of storage temperatures. We would like to point out that the stability of drugs/drug metabolites at body temperature may affect the detection window for select drugs that deposit in the meconium several weeks prior to birth. A marked decrease in the concentrations of 6-AM, nordiazepam, temazepam and oxazepam over a 2 week period at body temperature found by this study strongly suggests that these drugs may not be detectable by meconium if the mother uses the drugs more than 2 weeks before delivery, and the use of meconium for drug detection might underestimate the prevalence of these drug uses in pregnant women. Furthermore, repeat testing of meconium that occurs several weeks after initial testing may not yield equivalent results. Other analytical biomarkers might be necessary to determine prenatal drug exposure to these unstable analytes. There are limitations to our study and the associated results. First, this study was performed largely with spiked samples. It was not possible to assure that the incorporation of drug analytes in spiked samples was equivalent to the incorporation of drug analytes in authentic samples. We also did not include glucuronidated spikes, which would more accurately reflect the presence of conjugated drug analytes in authentic samples. Because our assays were not designed to specifically detect glucuronidated metabolites, we do not know the stability of glucuronidated metabolites, nor do we know the prevalence of these or other conjugated metabolites. Pre-analytical hydrolysis was performed for the benzodiazepine study only because it is part of routine testing of authentic samples. Second, the ph of the meconium was not tested. As such, it is not clear whether ph contributed to the stability of instability of specific drug analytes. Third, the instability of some drug analytes shown in this study offers one possible explanation for unexpected negative results, but cannot reflect the physiological stability exactly. Even the results from authentic samples cannot be translated to in-vivo stability, because the storage conditions of this study were in vitro. Lastly, imprecision of quantitation in meconium is higher than observed with other, less complex specimen types. This imprecision is particularly high (~30%) for m-hydroxybenzoylecgonine, for which isotopically labeled IS is not available. In our laboratory, this analyte is reported qualitatively, without any quantitative value. Thus, the routine reporting for this analyte is different than how the data were represented in this study, in order to capture stability trends. Conclusion In this study, we investigated the stability of 21 cocaine, opioid and benzodiazepine analytes in meconium at three storage temperatures. 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