Total Morphine Stability in Urine Specimens Stored under Various Conditions

Transcription

1 Total Morphine Stability in Urine Specimens Stored under Various Conditions Ber-Lin Chang, Min-Kun Huang, and Yu-Ying Tsai Division of Drug Chemistry, National Laboratories of Foods and Drugs, Department of Health, Executive Yuan, Kuen Yang Street, Nankang, Taipei, Taiwan Abstract The stability of total morphine in urine stored under various conditions was studied using control and experimental specimens. Samples in the control group were prepared using drug-free urine spiked with morphine at three concentration levels (300, 1000, and 2500 ng/ml), each with the ph adjusted to 5.5, 6.5, and 7.5. Samples in the experimental group came from 20 alleged heroin addicts (provided by Taipei Municipal Psychiatric Hospital). Samples in both groups were divided into two categories--one with and one without the precipitate (formed at 0~ removed. Samples in each of these two categories were further divided into two sub-groups--one with and one without sodium azide (0.05%) added. Total morphine contents in these samples were first determined by gas chromatography--mass spectrometry prior to storage and at 6, 12, 18, and 24 months following storage at -20, 4, 25, and 35~ Effects of sample treatment (azide addition and precipitate removal), ph, and storage temperature and length were evaluated by examining the percentage of total morphine remaining at the four time intervals following the initial determination. Major findings were as follows: (1) total morphine decomposition was minimal when stored for 12 months at -20~ which is a common current practice; (2) samples with lower initial sample ph had slower total morphine decomposition rates; and (3) azide addition appeared to have no detectable effect, whereas precipitate removal appeared to marginally reduce the decomposition rate, especially for samples with lower ph. Introduction Morphine and heroin are among the most commonly abused drugs in Taiwan. Reanalyses of total morphine in positive urine samples that have been stored for an extended period of time are often requested. When total morphine concentrations reported by the original and the repeated analyses differ significantly, interested parties often question the validity of analytical results and the effects of storage conditions on total morphine stability. There have been a few studies on this subject (1-3); many factors, however, have not been fully addressed. The purpose of this study is to contribute to the literature database on the effects of sample treatments (azide addition and precipitate removal), ph, and storage temperature and length on the stability of total morphine in urine samples. Materials and Methods Chemicals and reagents Drug-free urine (blank urine) was purchased from Bio-Rad (Anaheim, CA). Morphine solution (].0 mg/ml)and morphined:~ solution (].0 mg/ml) were purchased from Radian International (Austin, TX). ~LMethyl-lV-trimethylsityltrifluoroacetamide (MSTFA) was purchased from Aldrich Chemical (Milwaukee, WI). The ISOLUTE TM solid-phase extraction (SPE) columns (Confirm HCX 130 rag/3 ml) were purchased from International Sorbent Technology (Hengoed, Mid Glamorgan, U.K.). Twenty morphine-positive urine samples were obtained from Taipei Municipal Psychiatric Hospital (Taipei, Taiwan). All other chemicals and solvents were high-performance liquid chromatography grade and obtained from commercial sources. Sample preparation, storage conditions, and total morphine quantitation Preparation of samples in the control group. Drug-free urine (1800 ml) was divided into two 900-mL portions. One portion was placed in a refrigerator (0~ for approximately two days when the precipitation process appeared to have completed. Precipitate was removed by a centrifugation and filtration process. The resulting filtrate (approximately 900 ml) was set to regain the ambient temperature. Each of the two 900-mL portions of drug-free urine (one with precipitate removed and one without) was divided into two equal parts, and NaN3 (0.05%) was added to one portion in each group. 442 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 Each of the four 450-mL portions was divided into three 150-mL sub-parts into which morphine was added at one of three different concentration levels (300, 1000, and 2500 ng/ml). Each 150-mL sub-part was divided into three 50-mL aliquots, on which ph was adjusted (with phosphate buffer) to 5.5, 6.5, or 7.5. Control samples thus prepared were pipetted (1-mL aliquots) into 3-mL test tubes (Pyrex glass) and properly capped and labeled. One tube was randomly selected from each group and analyzed by gas chromatography-mass spectrometry (GC-MS) for total morphine concentration. Preparation of samples in the experimental group. A 210-mL portion was taken from each of the 20 morphine-positive urine samples obtained from Taipei Municipal Psychiatric Hospital. The ph of these samples was first measured, then divided into two portions (110 and 100 ml). One 110-mL portion was placed in a refrigerator (0~ to remove the precipitate following the same procedure used to prepare the corresponding samples in the control group. Procedures adapted for the preparation of the samples in the control group were followed to prepare respective samples in the experimental group. One tube from each group was also randomly selected and analyzed by GC-MS for total morphine concentration. Storage. A number of tubes from each category of samples were stored under four different temperature conditions: -20~ freezer, 4~ refrigerator, room temperature (25~ and 35~ incubator. Frequency ofquantitation. One sample from each category was reanalyzed for its total morphine concentration at 6, 12, 18, and 24 months after storage. Sample preparation for GC-MS analysis. To 1-mL urine samples were added 1 ml 0.4 ~g/ml morphine-d3 solution and 0.5 ml concentrated hydrochloric acid. Tubes were sealed and the samples hydrolyzed at 121~ for 15 rain. Tubes were cooled to room temperature, and 2 rnl 2.0M Tris buffer (ph 8.1) was added. KHCO3-containing 10M KOH solution (1.2 ml) was added to adjust sample ph to the range. The 2.0M Tris buffer (ph 8.1) was prepared by dissolving tris(hydroxymethyl) methylamine (24.2 g) in 90 ml water, and the ph was adjusted to 8.1 ( 0.1) using 1.0 M hydrochloric acid. Distilled water was added for a final volume of 100 ml. Extraction and derivatization. SPE was performed using ISOLUTE SPE columns following the procedures provided by the manufacturer (4). Extracts were evaporated to dryness, then derivatized with the addition of 100 IJL MSTFA (5), vortex mixed for 20 s, and kept for 10 rain at 90~ in a heating block. GC-MS instrumentation. GC-MS analysis was performed using a 5890 series II HP GC interfaced to a 5970 series HP mass selective detector (MSD, Hewlett-Packard, Palo Alto, CA). Separation was achieved with a HP5-MS capillary column (25 m x 0.25-mm i.d., Jm film thickness, Hewlett- Packard, Wilmington, DE). The MSD was operated in the selected ion monitoring mode. A Hewlett-Packard Laser Jet 4P printer, 7673 automatic sampler, and color display monitor were used in conjunction with ChemStation software (Version A3) for data analysis. An aliquot (1 I~L) of the derivatization product was injected using splitless mode with the injector maintained at 260~ and the GC-MS interface at 280~ The chromatography system was operated under the following conditions: helium carrier gas head pressure, 10 psi; column initial temperature, 200~ for 1 min; programming, 10~ to 280~ then held for 5 rain. The ions monitored were as follows: morphine, 429, 414, 401 and morphine-d3, 432, 417, 404. The molecular ions from the derivatized morphine and morphine-d3 at m/z 429 and 432, respectively, were used for quantitation. The ion ratios of the confirming ions were required to be within 20% of the same ion ratios of the calibration standard. Empirical LOD/LOQ determination (6). Series of standard solutions with successively lower morphine concentrations were prepared by making 1:4, 1:8, 1:16, and 1:32 dilutions of the 300-ng/mL standard solution with drug-free urine. These standards were analyzed 20 times over a three-week period. Assay LOD is defined as the concentration at which all standard GC-MS acceptance criteria (retention time within 2% and ion intensity ratios within 20% of the calibrator) are met at least 90% of the time. Standard acceptable chromatography criteria, such as peak symmetry and resolution, were also observed. Assay LOQ was defined as the concentration at which all acceptance criteria were met with the observed quantitative value within 20% of the target concentration. Limit oflinearity (7). The lower end of the assay linearity was set at the assay LOQ. The upper end of the linear range was arbitrarily set at 4500 ng/ml, on which repeated experiments were found to produce observed values well within 20% of the targeted values. Reproducibility studies. Assay between-run reproducibility was determined based on the results of 10 separate runs of 100- ng/ml morphine urine standards. Assay within-run reproducibility was based on the analysis of ng/mL morphine urine standards in one batch. Recovery study. Recovery studies were performed at six concentration levels (0.1, 0.3, 1.0, and 3.0 IJg/mL of morphine). One set of these standard solutions was processed through the extraction stage without the internal standard. Internal standard aliquots were then added to the resulting extraction products and to a second set of standards containing the same amount of morphine equivalent to the amount used in the first set. Both sets were then processed (in parallel) through the derivatization and the GC-MS analysis stages. Recoveries were calculated by dividing the amounts of the analyte observed in the first set by that observed in the second set. All recovery results are the means of three determinations. Results Summarized in Table I are some important analytical parameters established for the GC-MS protocol used in this study. This information provides a basis to evaluate the validity of analytical results derived from samples included in this study. Total morphine concentrations in treated and untreated control and experimental samples were determined at the beginning and four time intervals (6, 12, 18, and 24 months) following the storage under four different temperatures. Storage 443

3 temperatures were -20, 4, 25, and 35~ Sample treatments included ph adjustment (for control samples only), azide addition, precipitate removal, and both azide addition and precipitate removal. Shown in Table II are exemplary numerical data of total morphine concentrations and percentage changes following 12 and 24 months of storage at four temperatures. These numerical data are informative; however, because random errors are inadvertently imbedded in all determinations, it is difficult to evaluate total morphine stability relying on visual comparisons of individual numerical data. Instead, statistical trends of these data are used as the basis for total morphine stability evaluation. For example, ph effect on total morphine stability (in untreated experimental samples stored at 4~ is effec- tively demonstrated by plotting the percent of total morphine remained (after 24 months storage) against the sample's initial ph (Figure 1). Tables III-VII provide statistical information concerning the effects of storage conditions on total morphine stability. Specifically, percent of total morphine remained is plotted against time lapse (following original storage) when the determination was made. The resulting regression equations are tabulated for evaluation. Regression equations of total morphine remained (in percent) versus time of determination (0, 6, 12, 18, and y = x Table I. GC-MS Assay Parameters Parameter Limit of detection Limit of quantitation Linear range Established values 75 ng/ml 75 ng/ml ng/ml Reproducibility (mean; SD; n) Within-run (300 ng/ml) 300; 6.3; 10 Between-run (1 O0 ng/ml) 100; 7.2; 10 Recovery (mean; SD; n) 100 ng/ml 85%; 3.8; ng/ml 85%; 1.5; ng/ml 90%; 2.5; ng/ml 95%; 1.6; 3 O O Q. IOO ~ t m mm i i i h i i i Sample ph Figure 1. Effect of sample ph on total morphine stability (untreated experimental sample stored for 24 months at 4~ Table II. Total Morphine Concentrations and Percent Changes in Untreated Experimental Urine Samples after Storage for 12 and 24 Months After 12-month storage at -20~ After 24-month storage at -20~ Initial Initial conc. Sample ph (ng/ml) Conc. % Change Conc. % Change 4~ 25~ 35~ A , , , B A C D ,621 34, , E F G H [ ,288 63, , ] J K L ,077 46, , M N ,485 15, , O ,202 19, , P ,101 17, , Q R S ,923 28, , T ,

4 months) of control samples (treated and untreated and storage under different temperatures) that were adjusted to ph 5.5, 6.5, and 7.5 are listed in Tables III, IV, and V, respectively. Shown in Table VI are parallel information for experimental samples, which did not include ph adjustments. Regression equations listed in Table VII characterize the relationship between sample ph and the percentage of total morphine remained after 24 months of storage at four temperatures. Data shown in the second row (4~ of the first column (Not treated) is the same as the one shown in Figure 1. The slopes of the equations shown in Tables III-VII are adapted as indications of the extent that total morphine stability is affected by the parameters studied. These data are further discussed in the next section. Discussion Storage temperature Numerical data shown in Table II clearly indicate that storage temperature is the dominating factor on total morphine stability. It is comforting to note that the widely practiced storage of samples at -20~ for 12 months results Table III. Total Morphine Stability in Control Urine Samples at ph 5.5--Effects of Initial Total Morphine Concentration, Storage Temperature, and Treatments* Conc. NaN3 added and (ng/m/) Temp. Not treated NaN 3 added Precipitate removed precipitate removed ~ y = -3.78x * y = -2.19x + 104* y = 0.60x t y = x + 105* 4~ y = -8.39x y = -3.62x y = -0.29x y = -1.75x ~ y = -6.28x y = -4.53x y = -3.26x y = x ~ y = -12.6x y = -7.75x y = x y = -6.06x ~ y = -2.85x + 108* y = -1.75x t y = -0.89x * y = 1.58x * 4~ y = 2.76x * y = -3.40x y = -3.29x y = -0.45x ~ y =-4.63x y = -3.12x y = -3.11x y = -2.03x ~ y = -7.54x y = -10.6x y = -8.34x y = -6.39x ~ y= 0.24x t y= 1.85x + 102' y= 1.62x + 101' y= ~.26x t 4~ y=-2.29x 110 y=-3.76x y=-1.76x y=-1.61x ~ y=-11.0x y=-6.84x y=-4.94x+ 109 y=-7.33x ~ y=-14.9x y=-10.7x y=-6.01x y =-10.6x * y, percentage of total morphine remained and x, time in month. Variations in total morphine decomposition rates are too small for comparison with the corresponding data shown in Tables IV and V. * Outlier data comparing to corresponding data shown in Tables IV and V. Table IV. Total Morphine Stability in Control Urine Samples at ph 6.5--Effects of Initial Total Morphine Concentration, Storage Temperature, and Treatments* Conc. NaN 3 added and (ng/ml) Temp. Not treated NaN 3 added Precipitate removed precipitate removed ~ y = -4.12x + 108* y = -5.35x t y = -4.14x t y = -1.63x * 4~ y=-7.47x + 111' y=-4.38x y=-4.95x y =-2.41x ~ y = -4.59x * y = -4.66x y = -2.85x * y = -4.90x ~ y =-23.2x y=-16.6x y=-12.0x y=-9.83x ~ y=-2.12x + 105* y=-0.78x t y=-0.79x + 103' y= 1.53x t 4~ y=-6.81x y=-4.56x y=-8.31x ' y=-4.71x ~ y = -7.59x y = -5.24x y = -3.47x y = -6.63x ~ y=-12.7x y=-9.03x y=-10.6x y=-10.3x ~ y= 0.30x t y=-0.52x + 107' y=-0.14x + 105* y= 0.75x+ 104* 4~ y=-5.83x y=-o.98x + 106' y=-6.02x y=-3.18x ~ y = -9.57x + 120* y = -7.60x y = x y = -1.48x + 106' 35~ y=-14.6x y=-13.9x y=-13.0x+ 120 y=-15.8x * y, percentage of total morphine remained and x, time in month. t Variations in total morphine decomposition rates are too small for comparison with the corresponding data shown in Tables III and V. * Outlier data comparing to corresponding data shown in Tables III and V. 445

5 in non-detectable or minimal total morphine decomposition. The extent of total morphine decomposition at higher storage temperatures are reflected by the higher negative slopes of the equations listed in Tables III-V (control samples) and VI (experimental samples). Initial sample ph Figure 1 indisputably displays improved total morphine stability for samples with lower initial ph. This plot is the graphic presentation of one equation shown in Table VII (second row, first column; untreated experimental samples after 24-month storage at 4~ With the exception of samples stored at -20~ all data summarized in Table VII (representing experimental samples that were treated and untreated and stored under different temperatures) display the same trend on the effect of ph. (With no significant decomposition, ph effect on samples stored under -20~ is not apparent.) This ph effect is also evident in control samples when corn- Table V. Total Morphine Stability in Control Urine Samples at ph 7.5--Effects of Initial Total Morphine Concentration, Storage Temperature, and Treatments* Conc. NaN3 added and (ng/ml) Temp. Not treated NaN 3 added Precipitate removed precipitate removed ~ y = -2.53x + 103t y = -1.64x t y = -3.56x t y = -3.43x * 4~ y=-11.2x+ 113 y= -9.17x y=-9.82x y= -11.0x ~ y=-16.5x y=-16.1x y=-14.8x y=-12.3x ~ y=-24.1x y=-24.9x+ 119 y=-25.4x y= -24.7x ~ y = -0.36x t y = -I.29x t y = -0.69x + 101t y = x t 4~ y = -10.9x y = -11.8x y = -6.46x y = -6.06x ~ y = x + 108* y = -7.45x y = -17.3x y = -9.99x ~ y=-17.0x y =-19.5x y=-15.0x y=-16.4x ~ y = -1.37x t y = -0.45x t y = -0.58x t y = -1.35x t 4~ y=-ll.0x+ 117 y= -6.77x y=-8.41x+ 114 y=-11.2x ~ y = -11.8x y = -10.8x y = x y = -11.8x ~ y=-i 7.6x y=-20.3x y=-17.7x y=-19.2x * y, percentage of total morphine remaining and ".,, time in month. * Variations in total morphine de(omposiliiin rates are too small for (omparison with the corresponding data shown in Tables [11 and IV, ()utlk'r data ( onlf),lrin ~ [o (orresponding data shown in labk's III and IV. Table Vl. Total Morphine Stability in Experimental Urine Samples--Effects of Storage Temperature and Treatments* NaN 3 added and Temp. Not treated NaN 3 added Precipitate removed precipitate removed -20~ y=-4.47x y=-4.73x y= 2.85x ~ y=-10.8x y=-10.0x y=-7.49x ~ y=-18.4x y=-17.6x y=-14.3x ~ y=-lg.7x+ 116 y=-20.5x y=-18.3x /= -3.64x = -7.95x /=-15.2x x+ 118 y, per( enlage of total n/orphin{' remainir]g; x, lime in monlhi Table VII. Total Morphine Stability in Experimental Urine Samples--Effects of Initial ph, Storage Temperature, and Treatments* NaN 3 added and Temp. Not treated NaN 3 added Precipitate removed precipitate removed -20~ y = -0.83x y = -1.79x y = -6.18x y =-5.46x ~ y = -17.4x y = -13.8x y = -15.4x y = -17.6x ~ y=-13.3x y=-15.1x y=-18.6x y=-18.2x ~ y=-ll.6x+ 107 y=-10.3x y=-21.7x+ 196 y=-18.9x+ 174 * y, percentage of total morphine remained after 24-month storage and x, sample initial ph. 446

6 paring corresponding equations listed in Tables III (ph 5.5), IV (ph 6.5), and V (ph 7.5). There are, however, two exceptions: (1) total morphine decomposition rates for control samples stored under -20~ are minimal (cells in Tables III-V that were footnoted with an asterisk), thus, the ph effect is not apparent and (2) data in nine (those footnoted with a dagger) out of the remaining 108 cells are not consistent with the stated ph effect. These are probably results of experimental errors. Sample treatment Sample treatments, other than ph adjustment (i.e., azide addition and precipitate removal), appear to have only a minor or non-detectible effect on total morphine stability. These effects are most apparent for control samples with low concentrations (300 ng/ml) that were stored at 4, 25, and 35~ (see Tables III-V). Precipitate removal appears to have some effect in slowing down the decomposition rate of total morphine in the experimental samples, especially those with a lower initial ph (see Table VI). Initial concentration For the control group, the percent of total morphine remained after storage appears to be lower in samples where the initial total morphine concentration is low (300 ng/ml). This is, however, not apparent for samples in the experimental group. Apparently, other individual sample characteristics are more dominant factors. Conclusions Data generated by this study provide the following conclusions: (1) storage of samples for 12 months under -20~ - a common current practice--results in minimal or non-detectable total morphine decomposition; (2) improved total morphine stability is observed in samples with lower initial sample ph; and (3) sample treatments, such as azide addition and precipitate removal, appear to have no or minimal effect on total morphine stability. Acknowledgments The authors are grateful to Department of Health, Executive Yuan, Republic of China for supporting this research; the research project numbers are DOH86TD148 and DOH87TD143. The authors are also grateful to Professor Ray H. Liu of the University of Alabama at Birmingham (Birmingham, AL) for helpful discussion and assistance in the preparation of this manuscript and to Taipei Municipal Psychiatric Hospital (Taipei, Taiwan) for providing morphine-positive urine samples. References 1. B.D. Paul, R.M. McKinley, J.K. Walsh, Jr., T.S. Jamir, and M.R. Past. Effect of freezing on the concentration of drugs of abuse in urine. J. Anal. Toxicol. 17: (1993). 2. S. Dugan, S. Bogema, R.W. Schwartz, and N.T. Lappas. Stability of drugs of abuse in urine samples stored at -20~ J. Anal. Toxicol. 18: (1994). 3. D.L. Lin, H. Liu, and C.Y. Chen. Storage temperature effect on the stability of morphine and codeine in urine. J. Anal. Toxicol. 19: (1995). 4. Isolute TM SPE Application note, Extraction of codeine and morphine from urine, IST 1033, 1994, International Sorbent Technology LTD., IST House, Duffryn Industrial Estate, Ystrad Nynach, Hengoed, Mid Glamorgan, U.K. 5. Z. Liu, P. Lafolie, and O. Beck. Evaluation of analytical procedures for urinary codeine and morphine measurements. J. Anal. Toxicol. 18" (1994). 6. D.A. Armbruster, M.D. Tillman, and L.M. Hubbs. Limit of detection (LOD)/limit of quantitation: Comparison of the empirical and statistical methods exemplified with GC/MS assays of abused drugs. Clin. Chem. 40" (1994). 7. S.B. Needleman and R.W. Romberg. Limit of linearity and detection for some drugs of abused. J. Anal. Toxicol. 14:34-38 (1990). Manuscript received October 19, 1999; revision received January 10,