Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites

Transcription

1 Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites Ping Wang1, 2, Judith A. Stone 1,2, Katherine H. Chen 1,2, Susan F. Gross 1,2, Christine A. Hailer1,2,% and Alan H.B. Wu 1,2,* 1Clinical Laboratories, San Francisco General Hospital, San Francisco, California and Departments of 2Laboratory Medicine and 3Medicine, University of California at San Francisco, San Francisco, California I Abstract I Confirmation of opioids in urine samples of clinical patients requires liberation of opioids from their glucuronide conjugates. Both acid hydrolysis and enzyme hydrolysis using 13-glucuronidase from various sources have been reported, with the latter approach prevailing in most clinical toxicology laboratories. The goal of this study was to compare the efficiency of acid versus different enzyme hydrolysis methods in recovering morphine and common semisynthetic opioids from glucuronide standards and 78 patient urine samples that were screened positive for opioids as a class. Specimens were analyzed with a validated gas chromatography-mass spectrometry (GC-MS) procedure. With the exception of oxycodone, the results indicated that the majority of opioids tested were extensively glucuronideconjugated in urine. Significantly, acid hydrolysis liberated > 90% of morphine and hydromorphone from their glucuronide standards but enzyme hydrolysis had lower and variable efficiency, depending on the opiate type and the enzyme source. In patient specimens, much higher concentrations of free codeine, morphine, hydromorphone, and oxymorphone were obtained with acid hydrolysis than with various enzyme methods. Incomplete hydrolysis using 13-glucuronidase could lead to false-negative results for many opioids when urine is tested for drugs of abuse. We conclude that acid hydrolysis is the method of choice for GC-MS confirmation of urine opioids. Introduction Opioids are commonly prescribed to treat patients with short-term and chronic pain. A trend in recent years is that many prescription opioids are being increasingly diverted and abused by all age groups. According to the report from the Community Epidemiology Working Group of the National Institute on Drug Abuse, increases have been observed in many U.S. cities in sales and diversions, treatment admissions, poison center calls, and drug-related deaths for opioids other * Author to whom correspondence should be addressed: Alan H.B. Wu, San Francisco General Hospital, I001 Potrero Avenue, Rm 2M27, San Francisco, CA 941 I0, wualan@labmed2,ucsf.edu. than heroin (1). Amidst the controversy about appropriate conditions of opiate prescription, it is important to monitor the use of opioids in patients on opioid regimens. Urine testing of clinical patients for opioids provides important information in assessing patient compliance. Therefore, the ability of the clinical laboratory to accurately detect and differentiate various opioids is of clinical and forensic significance. Opioids are excreted in the urine as both the free and glucuronide-conjugated forms. Clinical screening of urine opioids is usually done by immunoassay and confirmation is achieved by gas chromatography coupled with mass spectrometry (GC-MS). For GC-MS confirmation, glucuronide conjugates of opioids are first hydrolyzed to convert opioids into their free forms. Free drugs are then extracted and derivatized before they are subjected to GC-MS analysis. Both acid hydrolysis and enzyme hydrolysis using [3-glucuronidase from different sources (e.g., Patella vulgate, Helix pomatia, Escherichia cell) have been reported, with the latter approach primarily used in both the literature and in most clinical laboratories (2-10). There are conflicting reports regarding which hydrolysis method yields higher recoveries of codeine and morphine. Some claim that acid has a lower efficiency in hydrolyzing codeine-6-glucuronide (7) and morphine-3-glucuronide (8) compared to [3-glucuronidase. However, most analysts agree that both acid and ~-glucuronidase can efficiently hydrolyze > 90% of morphine- 3-glucuronide when reaction conditions are optimized (3-5), although complete hydrolysis by ]3-glucuronidase may require incubation for as long as 16 h. Much less effort has been devoted to characterizing the efficiencies of different hydrolysis methods for other opiate glucuronides, including morphine-6-glucuronide. Romberg et al. (4) and Hackett etal. (5) reported that the hydrolysis rates of morphine-6-glucuronide and codeine-6- glucuronide were much lower than that of morphine-3-glucuronide with both acid and enzyme methods. In these two studies, acid hydrolysis yielded higher recovery than the enzymatic method (using enzymes from either Helix pomatia or Escherichia coli) for both morphine-6-glucuronide and codeine-6-glucuronide. These results suggested that the hydrolysis rate can vary among different opiate conjugates, and hydrolysis protocols should be optimized for each opiate tested, not just for morphine-3-glucuronide. 570 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 There are no reported hydrolysis studies for confirmation testing of the 6-keto-opioids. Considering the different electrochemical environment of glucuronides in the 6-keto opiate conjugates (see Figure I for the structures of different opioids), it is very likely that the hydrolysis rates of these opioids differ from that of morphine-3-glucuronide. Nevertheless, most clinical laboratories assume that hydrolysis protocols using 9- glucuronidase optimized for morphine-3-glucuronide would also perform equally well for clinical samples containing the other opiate glucuronides. This presumption may lead to impaired ability of clinical laboratories to detect opioids other than morphine. Most of the hydrolysis studies in the literature were carried out using negative urines spiked with purified opiate-glucuronide standards. No detailed studies have been performed with patient urine samples to compare acid hydrolysis and enzyme hydrolysis, as well as 13-glucuronidases from different sources. Combie et al. (10) evaluated four [3-glucuronidases for hydrolysis of morphine glucuronide in equine urine samples after morphine sulfate was administered intravenously. They found that the enzyme from Patella vulgata had higher efficiency compared to those from Helix aspersa, Helixpomatia, and bovine liver, suggesting that the source of [3-glucuronidases can affect the hydrolysis performance. However, neither acid hydrolysis nor opioids other than morphine were included in their comparison. In this study, the recovery of both natural (codeine and morphine) and commonly prescribed semi-synthetic opioids (hydromorphone, oxycodone, and oxymorphone) from commercially available glucuronide standards and patient urine samples was compared by acid hydrolysis and enzyme hydrolysis. Although hydrocodone (Vicodin) is also a commonly prescribed opiate, it does not have any hydroxyl group for glucuronidation, and therefore was not tested here. Three commonly used 13-glucuronidases (Patella vulgata, Helix pomatia, and Escherichia coli) were tested. All patient samples were also extracted without hydrolysis to quantify unconjugated (free) drug concentrations. A r~c...o.~ D H3o~O0..~ B HO.~ E HO~ O~,V.J~'OH ""C H 3 o~n~ch3 C 0 ~N~cH3 Figure 1. Chemical structures of the opioids tested in this study: codeine (A), morphine (B), hydromorphone (C), oxycodone (D), and oxymorphone (E). The three carbons (3, 6, and 14) that hydroxyl groups may attach to, and thus glucuronidation may occur, are numbered in B. Methods Enzymes [~-Glucuronidase from Patella vulgata (catalog # G8132), Helixpomatia (catalog # G0751), and Escherichia coli (catalog # G7396) were obtained from Sigma Chemical (St. Louis, MO). Enzyme solutions were prepared at a concentration of 5000 U/mL in appropriate buffers. The buffers and phs used for each enzyme were as follows: P. vulgate in 1.0M acetate buffer, ph 5.0; H. pomatia in 1.0M acetate buffer, ph 5.0; and E. coli in 0.1M phosphate buffer, ph 6.8. Enzyme solutions were stored as aliquots at -20~ until use. Opiate glucuronide standards Morphine-6-glucuronide (catalog # M-096) and hydromorphone-3-glucuronide (catalog # H-051) were purchased from Cerilliant (Round Rock, TX). Drug-free human urine [catalog # CDF(L)] was obtained from Utak Laboratories (Valencia, CA). Twenty-five microliters of 100 lag/ml morphine-6-glucuronide or hydromorphone-3-glucuronide standard was added to 5 ml drug-free urine to prepare the glucuronide standard solution for analysis. Morphine-3-glucuronide was present at the concentration of 375 ng/ml in a custom-made lyophilized quality control material from Utak Laboratories and was reconstituted with reagent grade water. Two milliliters of each drug standard solution was analyzed. Mixed opiate internal standard (50 pl) was added to each sample to give a concentration of 300 ng/ml morphine-d3, 300 ng/ml codeine-d3, 300 ng/ml hydrocodone-d3, 300 ng/ml hydromorphone-d3, 100 ng/ml oxycodone-d3, and 100 ng/ml oxymorphone-d3 (stock solutions all obtained from Cerilliant). Two hundred microliters of methoxyamine hydrochloride (2%) in pyridine (catalog # 45950, Pierce Biotechnology, Rockford, IL) was then added to prevent enolization of hydrocodone and hydromorphone. Patient urine samples Urine samples containing different opioids were selected from a frozen collection of past patient specimens. Approval from the Institution Review Board of University of California San Francisco was obtained to use these samples for this study. All samples were tested positive using the CEDIA (Microgenics, Fremont, CA) on the ADVIA 1650 chemistry analyzer (Bayer Healthcare, Tarrytown, NY). Samples were identified by study numbers without patient information. The numbers of specimens tested for each opiate were as follows: 15 for morphine, 10 for codeine, 12 for hydromorphone, 20 for oxycodone, and 21 for oxymorphone. Two milliliters of each patient urine sample was analyzed. Internal standard and methoxyamine derivatizing reagent were added as described for glucuronide standards. Acid hydrolysis For measurement of total opioid concentrations, 2 ml of hydrochloric acid (ACS grade, %) was added to 2 ml of each sample followed by incubation in a heating block at 100 5~ for 90 min. Samples were then cooled to room temperature before ph was adjusted with ammonium hydroxide to be between 6.0 and 7.0. Biologic debris in each sample was pre- 571

3 cipitated by centrifugation. The supernatant was subjected to solid-phase extraction. For quantitation of free opioids in patient samples, 2 ml of 1.0M acetate buffer (ph 5.0) instead of hydrochloric acid was added to each sample. Enzyme hydrolysis Ten thousand units of [3-glucuronidase were added to each sample followed by incubation in a heating block at the optimal temperature for each enzyme for 2 h. The temperatures used for each enzyme were as follows: 65~ for P. vulgate, 60~ for H. pomatia, and 50~ fore. coil For all glucuronide standards treated by [3-glucuronidase from H. pomatia or E. coli, two incubation intervals were tested: 2 and 16 h. Sample ph was then adjusted, and samples were centrifuged as described for acid hydrolysis. Solid-phase extraction Clean Screen CSDAU columns (catalog # CSDAU206, United Chemical Technologies, Bristol, PA) were prepared by washing sequentially with methanol, water, and 0.1 mol/l phosphate buffer (ph 6.0). Supernatant from each sample was then slowly drawn through the column by vacuum. Columns were then washed sequentially with water, 0.1 mol/l acetate buffer (ph 4.5), and methanol. After the columns were dried under high vacuum for 5 rain, opioids were eluted with 3 ml of elution solvent (78:20:2 of methylene chloride/isopropanol/ammonium hydroxide). The extracts were evaporated to dryness with a stream of nitrogen at 37~ and derivatized by adding 50 I~L of ethyl acetate and 50 I~L of N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (catalog # , Regis Technologies, Morton Grove, IL), followed by incubation at 65 _+ 5~ for 20 min. GC-MS All samples were analyzed with an Agilent model 5973 GC-MS (Agilent Technologies, Palo Alto, CA). The assay was calibrated each run with a urine calibrator containing 300 ng/ml each of codeine, morphine, hydrocodone, and hydromorphone, and 100 ng/ml each of oxycodone and oxymorphone. Calibration data were fit by one point, linear regression without forcing through the origin and with equal weighting. Quantitation of the opioids in each sample was achieved by determining the drug-to-internal standard ratio at the following mass-to-charge ratios: 371/374 for codeine, 429/432 for morphine, 386/389 for hydromorphone, 416/419 for oxycodone, and 474/477 for oxymorphone. Qualifying ions at the following mass-to-charge ratios were monitored for different opioids: 343 and 234 for codeine, 414 and 401 for morphine, 355 and 371 for hydromorphone, 401 and 229 for oxycodone, and 459 and 287 for oxymorphone. Qualifying ion ratios were required to be within specified ranges around the values found for the calibrator on each run (_+ 5% absolute for ions with < 20% abundance, + 20% relative for ions with 20-50% abundance, and + 10% absolute for ions with > 50% abundance). The linear ranges of the assay were ng/ml for oxycodone, oxymorphone, and morphine and ng/ml for all other opioids. The limits of quantitation were 50 ng/ml for oxycodone and oxymorphone, and 100 ng/ml for all other opioids. The intra- and interday CVs for quality control materials at concentrations 25% below (negative) and 25% above (positive) the calibrator for all opioids were < 13%. The intraday CV for patient specimens was < 10%. Results Hydrolysis of opiate glucuronide standards The efficiency of hydrolysis methods in recovering morphine and hydromorphone from three commercially available glucuronide standards was compared. The percentage of recovery was calculated by determining the ratio between the GC-MS quantitation and the nominal value of the standard solution. Each standard was analyzed with n > 3, at at least two different concentrations. Means of all results and standard deviation from the mean were listed in Table I. As shown, acid hydrolysis using concentrated HC1 recovered % of morphine or hydromorphone from their respective glucuronide conjugates. The [3-glucuronidase from P. vulgata hydrolyzed 94% of morphine-3-glucuronide, only 12% of morphine-6-glucuronide, and 49% of hydromorphone-3-glucuronide. The enzyme from H. pomatia had lower efficiencies: 33% of morphine-3-glucuronide and 10% of hydromorphone- 3-glucuronide were recovered after a 2-h incubation; when the length of incubation was increased to 16 h, the hydrolysis percentages increased to 50% and 20%, respectively. No morphine- 6-glucuronide was recovered by the H. pomatia enzyme, even after a 16-h incubation. Under the experiment conditions used in this study, the E. coli enzyme was not able to hydrolyze any of the three opiate glucuronide conjugates. Table I. Percent of Recovery of Opioids from Commercial Opioid Glucuronide Standards by Different Hydrolysis Methods H. pomatia E. coil Acid P. vulgata 2 h 16 h 2 h 16 h Standard Mean SD* Mean SD Mean SD Mean SD Mean SD Mean SD Morphine-3-glucuronide (%) 100 Morphine-6-glucuronide (%) 98 Hydromorphone-3-glucuronide (%) * SD = standard deviation from the mean. 572

4 Hydrolysis of different opioid conjugates in urine samples of clinical patients Both the acid and enzyme hydrolysis methods were further tested using patient urine samples containing different opioids (codeine, morphine, hydromorphone, oxycodone and oxymorphone). Each opioid was analyzed in different patient samples at a wide range of concentrations, from < 100 ng/ml to > 100,000 ng/ml (with dilutions). Means of recovery and standard deviations from different patient samples were listed in Table II. To quantify how much of each opioid was present as glucuronide conjugates in the urine after human metabolism, each patient sample was also analyzed without any hydrolysis treatment. All enzyme hydrolysis experiments were carried out after an incubation period of 2 hours. The hydrolysis method generating the highest free drug concentrations was assigned as 100% hydrolyzed. This occurred under acid hydrolysis conditions in all cases. As shown in Table II, less than 10% of codeine, morphine, hydromorphone, or oxymorphone was recovered from patient urines without any hydrolysis treatment, indicating that the majority (> 90%) of these opioids existed in urine as glucuronide conjugates after extensive metabolism. The only exception was oxycodone. As can be seen in Table II, 99% of oxycodone could be detected without hydrolysis, suggesting the majority of oxycodone in urine was present in its unconjugated or free form. As a consequence, all four hydrolysis methods yielded the same recovery (- 100%) for oxycodone, because no glucuronide hydrolysis was needed. For all other opioids, acid hydrolysis gave the highest recoveries. Up to 5 times higher concentrations of free drugs were obtained with the acid method than with the R vulgata enzyme method. Compared to the H. pomatia enzyme method, acid hydrolysis yielded up to 10 times higher concentrations of free drugs. Both of these enzymes had the highest efficiency in converting morphine glucuronides, followed by oxymorphone and hydromorphone glucuronides, with the poorest yield for codeine glucuronides. The [~-glucuronidase from E. coli had virtually no activity for any of the opioids tested. Discussion In this study, four hydrolysis methods were compared for their effectiveness in converting opiate glucuronides to free drugs to facilitate clinical opiate confirmation using GC-MS. These methods included acid hydrolysis using concentrated HC1 and enzyme hydrolysis using [3-glucuronidase from three sources: R vulgata, H. pomatia, and E. coil Prescription opioids tested were morphine, codeine, hydromorphone, oxycodone, and oxymorphone. The experiments using commercially available glucuronide standards and urine samples of clinical patients led to consistent conclusions: acid hydrolysis had higher effectiveness than any of the three enzyme methods; and 13-glucuronidases from three sources possessed variable degrees of activity for opioids, with the R vulgata enzyme better than the/t, pomatia enzyme, followed by the E. coli enzyme, which did not hydrolyze any opiate conjugates under the experiment conditions tested. The/-/. pomatia and the E. coli enzymes were not tested with 16-h incubation for patient urines, since the increased incubation length only gave slight improvements, if any, in the activities of these two enzymes for glucuronide standards (Table I). Incubation for as long as 16 h would be sub-optimal from the assay turnaround time standpoint in a clinical laboratory setting and therefore is not recommended as a means to increase free drug recovery. An interesting finding is that although the same protocols (buffer, ph, temperature) as described in two previous studies (4,5) were followed for hydrolysis using the H. pomatia and the E. coli enzymes, the percentages of morphine we recovered from morphine glucuronide were much lower than what were reported in the two previous studies. We repeated our experiments using two different batches of fresh enzyme preparations for each enzyme, and obtained similar results. These two batches were from the same and the only available lots of the manufacturer. One possible explanation is that we were using enzymes from lots different from those used in previous studies, and there are variations in enzyme activity between lots. We noticed that the manufacturer validates the activity of both enzymes by monitoring liberation of phenolphthalein from phenolphthalein glucuronide at 37~ for 30 rain. Because the enzyme activities were defined using assay, temperature, and length of incubation all different from the opioid hydrolysis assay, it is possible that the enzyme activities varied between lots for our assay. The results from this study indicate that acid hydrolysis might be the method of choice for urine opiate drug confirmation in clinical patients. Although it was reported that acid hydrolysis with incubation at high temperatures yielded higher background noises and more extraneous peaks than the enzyme method (2), this was not found to be a problem in the Table II. Percent of Recovery of Opioids from Patient Samples by Different Hydrolysis Methods Acid P. vulgata H. pomatia E. coil No Hydrolysis Opioid Mean SD* Mean SD Mean SD Mean SD Mean SD Codeine (%) Morphine (%) Hydromorphone (%) Oxycodone (%) Oxymorphone (%) * SD = standard deviation from the mean. 573

5 current study. In contrast, low recovery of opioids with the enzyme methods often resulted in distortion of peak shapes and disqualification of the qualifier ion ratios (Figure 2). Another disadvantage of the enzyme methods is that endogenous inhibitors (e.g., saccharo-l,4-1actone) present in some patient urine samples might interfere with hydrolysis (10,11). Also, as described in the previous paragraph, enzyme activity variation between different lots can cause significant result changes. In contrast, acid hydrolysis has the advantage of higher recovery, higher consistency and less interference, but has the disadvantage of greater time required in adjusting sample ph before the solid-phase extraction step. Incomplete hydrolysis using various 13-glucuronidase could lead to false negative results for codeine, morphine, hydromorphone and oxymorphone. For example, when one patient urine sample in this study was assayed for morphine (screened positive for 400 ng/ml opioids by immunoassay), we obtained morphine concentrations of 271 ng/ml using acid hydrolysis, and 0 ng/ml using [B-glucuronidase from P. vulgata. In the latter case, the morphine signal on GC-MS was so low that the qualifier ions were distorted and ion ratios were out of the acceptable ranges. Using the cutoff concentration of 100 ng/ml for morphine confirmation, this patient specimen would be confirmed positive for morphine using acid hydrolysis, but negative using enzyme hydrolysis. Similar scenarios occurred for codeine, hydromorphone, and oxymorphone in our study. Oxycodone detection might also be impaired because oxycodone is quickly metabolized to oxymorphone (12). Because enzyme hydrolysis is the dominant method used in current practice of most clinical toxicology laboratories, these results may help to explain the variance in the ability of clinical laboratories to detect opioids in patient specimens. Successful performance with CAP survey samples can be misleading, as opioids are present in CAP samples as free drugs (except for the morphine glucuronides). In a quantitative comparison study, we had patient urine samples measured at other reference laboratories that use enzyme hydrolysis for opioid confirmation and compared to our acid hydrolysis results. We also analyzed some CAP survey samples using acid hydrolysis and compared Codes Amount : Ion: (I0. 762) Io-~T." "3-43-~0. 761) Resp: I Resp: /21.7 ll Rnge//' 16,7-25.1,o:,o [ 1080,~ ,85 ng/ml Ion: 234 (i0.759) --] Resp: /50.2 ~ Rngeh ,4 i /I ' f... ] IOBO 11.~ i Codeine-TMS Amount : 8.14 n~/ml... "I~n:--3-7i---2-~i-0~-7-62)... T-Ion: 343"~I0,'802)... V-Ion: 234 (I0. 738) [ Resp: Resp: /99.8 ]Resp: /133.1 Rnge: / Rng~-~ tl.20 [ Figure 2. Examples of single ion monitoring profiles of codeine in a patient urine sample, obtained using either acid (upper panel) or P. vulgata enzyme hydrolysis (lower panel). Note that the response signal was much smaller in the lower panel; the qualifying ion peak shapes were distorted, and the ion ratios failed to fall within the specified ranges in the lower panel, resulting in the concentration of codeine obtained being unreliable. Journal of Analytical Toxicology, Vol. 30, October 2006 our results to the target values. We observed significant differences between our biases for CAP samples and patient sampies: for CAP samples the bias of our results ranged from -14% to 2%; for patient samples we consistently obtained higher values than other laboratories, with a bias of 25% to162% for hydromorphone and 34% to 52% for oxymorphone. The high positive bias for patient samples but not CAP samples may be because hydromorphone and oxymorphone are extensively glucuronide-conjugated only in the patient samples, and the acid hydrolysis method we use can recover more opioids. It was generally assumed that hydrolysis conditions using [5- glucuronidase optimized for morphine-3-glucuronide would also perform optimally for other opiate glucuronides. However, only up to 64% of morphine was successfully recovered from patient urines in this study using [B-glucuronidase (because of the presence of morphine-6-glucuronide in the urines), not to mention other opiate conjugates less readily hydrolyzed. Therefore, hydrolysis protocols optimized using only commercial morphine-3-gtucuronide standard should not be extrapolated to urines of clinical patients containing many different opiate conjugates. It has been shown in this study that smaller percentages of morphine-6-glucuronide were recovered with all hydrolysis methods, as compared to morphine-3-glucuronide (Table I). Data in Table II indicate that [5-glucuronidases from P. vulgata and H. pomatia were more efficient in hydrolyzing morphine glucuronides than codeine ghcuronides. These results suggest that the glucuronide group at carbon 6 is much less readily hydrolyzed than the one at carbon 3, which is consistent with previous literature reports (4,5). As suggested by Romberg and Lee (4), a possible explanation is that the glucuronic acid group of morphine-3-glucuronide is attached to an electron-rich phenolic position (see Figure 1B), and therefore would be easier to hydrolyze because electron resonance would stabilize the resuiting phenolate anion. In contrast, the glucuronic acid at the 6 position is bound to an alcoholic group and is more stable (4). The finding that the majority of oxycodone is present as unconjugated form after metabolism is an interesting observation. The only hydroxyl group of oxycodone is attached to carbon 14 as an alcoholic group (Figure 1D), which is quite stable as there are no electron-rich double bonds in the vicinity. This group is also possibly protected within the ring structure formed by five carbons and one nitrogen atom, making it less accessible to UDP-glucuronosyltransferase, the enzyme responsible for glucuronidation of the hydroxyl group. If the hydroxyl group at carbon 14 of oxycodone is not glucuronidated, it is reasonable to deduce that no glucuronic acid would react with the hydroxyl group at carbon 14 of oxymorphone either, leaving only the hydroxyl group at the 3 position of oxymorphone available for glucuronidation. Therefore, we can postulate that the majority of oxymorphone in the urine is present as oxymorphone-3-glucuronide, which shares the same structure with hydromorphone-3-glucuronide (the major form in which 574

6 hydromorphone is present in the urine), except for the hydroxyl group at position 14. It is thus not surprising that each hydrojysis method showed very similar efficiencies in recovering hydromorphone and oxymorphone from patient urines (Table II). Another observation from the data in Table II is that the recovery of morphine demonstrated the highest degree of variation among all opioids (see the standard deviations in Table II). Because a similar number of patient urines were analyzed for each opiate, this finding suggests that a more diverse metabolism pattern exists for morphine among individuals. This hypothesis needs to be further tested by studies involving larger numbers of patients and perhaps coupled with pharmacogenetic studies. It is known that morphine is mainly metabolized through glucuronidation by UDP-glucuronosyltransferase 2B7 (UGT2B7) (13) and N-demethylation by cytochrome P450s (CYP3A4 and CYP2C8) (14). Clinical studies have shown that metabolizing enzyme polymorphisms can lead to variation in metabolites and drug effects of opioids [e.g., codeine metabolism to morphine was significantly different in extensive and poor metabolizers (15,16)]. Therefore, a pharmacogenetic study correlating UGT2B7 and CYP3M genotypes with morphine metabolism phenotypes is likely to improve the understanding of our current observation. An alternative explanation of this diverse metabolism pattern is that morphine was administered by different routes in these patients, since it has been published that the percentage of morphine-6-glucuronide varies with the mode of administration (17). Morphine dosing history was not available to confirm or refute this possibility. Finally, although we have shown that acid hydrolysis can definitively liberate > 95% of morphine or hydromorphone from their glucuronide conjugates, its absolute efficiency in hydrolyzing codeine or oxymorphone glucuronides was not tested in this study because these standards are not commercially available. Therefore, the 100% efficiency stated for acid hydrolysis of codeine and oxymorphone is relative, based on the finding of lower recoveries with all other hydrolysis protocols. Conclusions Enzyme hydrolysis had variable effectiveness in converting opiate glucuronides to free drug forms, depending on the opiate type and the enzyme source, with oxycodone (unconjugated) > morphine > oxymorphone and hydromorphone > codeine, and ~-glucuronidase from P. vulgata > H. pomatia > E. coil Acid hydrolysis liberated > 90% of the opioids tested from the glucuronide standards. Much higher concentrations of free drugs were obtained from patient samples with acid hydrolysis than with various enzyme methods, except for oxycodone, which is present in the urine mostly in the unconjugated form. Use of different hydrolysis methods may underlie variance in the ability of clinical laboratories to detect opioids in patient specimens. Incomplete hydrolysis using the enzyme methods could lead to potential false-negative results for codeine, morphine, hydromorphone, and oxymorphone. Therefore, we propose that acid hydrolysis should be the method of choice for GC--MS confirmation of opioids in urine. References 1. Community Epidemiology Work Group, National Institute on Drug Abuse. Epidemiologic trends in drug abuse--advance Report, June 2005, March R. Meatherall. GC-MS confirmation of codeine, morphine, 6- acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine. J. Anal. Toxicol. 23: (1999). 3. T.A. lennison, E. Wozniak, G. Nelson, and EM. Urry. The quantitative conversion of morphine 3-~-D glucuronide to morphine using 13-glucuronidase obtained from Patella vulgata as compared to acid hydrolysis. J. Anal. Toxicol. 17: (1993). 4. R.W. Romberg and L. Lee. Comparison of the hydrolysis rates of morphine-3-glucuronide and morphine-6-glucuronide with acid and i3-glucuronidase. J. AnaL ToxicoL 19: (1995). 5. L.P. Hackett, L.J. Dusci, K.E Ilett, and G.M. Chiswell. Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther. Drug Monit. 24: (2002) 6. M. Cremese, A.H. Wu, G. Cassella, E. O'Connor, K. Rymut, and D.W. Hill. Improved GC/MS analysis of opioids with use of oxime- TMS derivatives. J. Forensic Sci. 43: (1998). 7. ET. Delbeke and M. Debackere. Influence of hydrolysis procedures on the urinary concentrations of codeine and morphine in relation to doping analysis. J. Pharm. Biomed. Anal. 11: (1993). 8. A. Solans, R. de la Torre, and J. Segura. Determination of morphine and codeine in urine by gas chromatography-mass spectrometry. J. Pharm. Biomed. Anal. 8: (1990). 9. L.A. Broussard, L.C. Presley, T. Pittman, R. Clouette, and G.H. Wimbish. Simultaneous identification and quantitation of codeine, morphine, hydrocodone, and hydromorphone in urine as trimethylsilyl and oxime derivatives by gas chromatography-mass spectrometry. Clin. Chem. 43: (1997). 10. J. Combie, J.W. Blake, T.E. Nugent, and T. Tobin. Morphine glucuronide hydrolysis: superiority of 13-glucuronidase from Patella vulgate. Clin. Chem. 28" (1982). 11. R.L. Bertholf, L.M. Sapp, and D.L. Pittman. Failure of 13-glucuronidases to hydrolyze exogenous morphine glucuronide. Clin. Chem. 37: (1991). 12. R.C. Baselt. Disposition of Toxic Drugs and Chemicals in Man, 7th ed. Chemical Toxicology Institute, Foster City, CA, 2004, pp B.L. Coffman, G.R. Rios, C.D. King, and T.R. Tephly. Human UGT2B7 catalyzes morphine glucuronidation. Drug Metab. Dispos. 25:1-4 (1997). 14. D. Projean, RE. Morin, T.M. Tu, and J. Ducharme. Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 s responsible for morphine N-demethylation in human liver microsomes. Xenobiotica 33: (2003). 15. K. Eckhardt, S. Li, S. Ammon, G. Sch~inzle, G. Mikus, and M. Eichelbaum. Same incidence of adverse drug events after codeine administration irrespective of the genetically determined differences in morphine formation. Pain 76:27-33 (1998). 16. E. Haffen, G. Paintaud, M. Berard, C. Masuyer, Y. Bechtel, and ER. Bechtel. On the assessment of drug metabolism by assays of codeine and its main metabolites. Ther. Drug Monit. 22: (2ooo). 17. P.K. Janicki, W.A.R. Erskine, and M.F.M. james. The route of prolonged morphine administration affects the pattern of its metabolites in the urine of chronically treated patients. Eur. J. Clin. Chem. Clin. Biochem. 29: (1991 ). 575