Journal of Analytical Toxicology, Vol. 31, November/December 2007
Determination of Illicit Drugs and their Metabolites in Human Urine by Liquid ChromatographyTandem Mass Spectrometry Including Relative Ion Intensity Criterion Marta Concheiro, Ana De Castro, Oscar Quintela, AngelinesCruz, and Manuel L6pez.Rivadulla" University of Santiagode Compostela, Forensic Toxicology Service, Spain
Abstract I A method, using 0.5 mL of urine, was developed for the simultaneous determination of ecgonine methyl ester, benzoylecgonine, morphine, codeine, 6-acetylmorphine, amphetamine, methamphetamine, 3,4methylenedioxyamphetamine (MDA), 3,4. methylenedioxymethamphetamine (MDMA), methadone, 2-ethylidene-l,5.dimethyl-3,3-diphenylpyrrolidine (EDDP), and d-lysergic acid diethylamide (LSD). The analysis was performed by liquid chromatography with tandem mass spectrometry, after solid.phase extraction in the presence of their deuterated analogues. Reversed-phase separation on an Atlantis dC18 column was achieved in 12.5 rain, under gradient conditions. The method was fully validated, including linearity (1-2000 pg/L for ecgonine methyl ester, benzoylecgonine, 6-acetylmorphine, methamphetamine, MDMA, and EDDP;2-2000 pg/L for morphine, codeine, MDA, and methadone; 2-1000 pg/L for amphetamine, and 0.2-100 pg/L for LSD;rz > 0.99); recovery (> 65%), within-day and betweenday precision, and accuracy (CV and MRE < 15%); limit of detection (0.1 pg/L for LSD, 0.5 pg/L for ecgonine methyl ester, benzoylecgonine, methamphetamine, MDMA, 6-monoacetylmorphine, and EDDP,and 1 pg/t for amphetamine, MDA, morphine, and methadone); quantitation (lowest level of the calibration curve); relative ion intensities, freeze-and-thaw stability, and matrix effect. The procedure showed to be sensitive and specific, and was applied to real casesand quality control samples from a quality control program.
Introduction Laboratory drugs of abuse testing have traditionally been based on urine, due to its concentrations of metabolites and/or parent drugs being usually higher than in another biological matrix such as blood or oral fluid, as well as being higher in the volume collected (20 mL). Depending on the half-life of the
drug, the excretion pattern, and the sensitivityof the analytical test, many drugs may be detected in urine for a few days to a week following the last use of the drug (1). According to this, urine is the selected biological matrix if a recent past use of a drug has to be proven, for example in pre-employment, random, return-to-duty, follow-up, sport drugs of abuse testing, or in drug-facilitated sexual assault cases (DFSA) wherein low drug levels have to be detected (2). In forensic laboratories, immunoassays are commonly used as screening techniques. According to the SOFT/AAFSGuidelines (3), this initial detection of drugs should be confirmed by a second technique based on a different chemical principle, like gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). GC-MS is still the most widely used method of reference, but LC coupled with MS or tandem mass spectrometry (MS-MS) is becoming increasingly important for the identification and quantification of analytes (4--6), especially for the more polar, thermolabile, or low-dosed drugs (7). These confirmatory methods should be fully validated and special attention should be paid to the compound identification criteria. Besides retention time and the presence of at least two multiple reaction monitoring (MRM) transitions or two selected ion monitoring (SIM) ions, another important identification criterion is the ion ratio or relative ion intensities. While its allowed variations are well established for GC-MS (8,9), further research should be done for LC-MS because of the differences in the fragmentation pattern obtained with different instruments. Fully validated methods for the determination of multiple illicit drugs have been published on oral fluid (10-13), on plasma (14-16), and on urine (17,18). In urine, the groups of compounds simultaneously determined were opiates and cocaine (18); opiates, cocaine and amphetamines (10,11,14,17); opiates, cocaine and methadone (12,16); and opiates, cocaine, methadone, and LSD (15). None of the authors has completely followedthe compound identification criteria (retention time, two MRM transitions or SIM ions, and relative ion intensi-
9Author to whom correspondenceshould be addressed.E-mail:
[email protected]. Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
573
Journal of Analytical Toxicology, Vol. 3 I, November/December2007
ties), except Maralikova et al. (16) for opiates, cocaine, and methadone. The method developedis the first fully validated LC-MS--MS method which includes relative ion intensity data for the simultaneous determination of morphine, 6-acetylmorphine (6AM), codeine, amphetamine, methamphetamine, MDA,MDMA, ecgonine methyl ester, benzoylecgonine, methadone, EDDP, and LSD in urine.
Experimental Apparatus The extraction procedure was performed using Gilson AspecXLautomated solid-phase extraction equipment (VilliersLe Bel, France). The HPLCsystem was a Waters Alliance2795 separation module with a Waters Alliance series column heater/cooler (Waters, Milford, MA). The detection was performed using a Quattro MicroTM API ESCI triple quadrupole mass spectrometer (Micromass, Waters) fitted with a Z-spray ion interface. The instrument was operated in electrospray positive ionization mode (ESI+). Data acquisition, peak integration, and calculation were interfaced to a computer workstation running MassLynxNT 4.1 and QuanLynx4.1 software.
Reagents Morphine, 6-acetylmorphine, codeine, d,l-amphetamine (A), d,l-methamphetamine (MA), d,I-MDA,d,I-MDMA,benzoylecgonine (BE), and internal standards (IS) morphine-d3, 6-monoacetyl-morphine-d3(6-AM-d3),codeine-d3,d,l-methamphetamine-d5 (MA-ds), d,I-MDMA-d5,and benzoylecgonine-d3 (BE-d3)were obtained from Lipomed (Arlesheim,Switzerland) in solid form. Ecgonine methyl ester (EME), LSD, ecgonine methyl ester-d3 (EME-d3) in acetonitrile at 1 mg/mL, LSD-d3 in acetonitrile at 100 rag/L, methadone, EDDP,methadone-d3, MDA-d5 in methanol at 1 g/L, amphetamine-ds (A-ds), and EDDP-d3 in methanol at 100 mg/L were obtained from Cerilliant (Round Rock, TX). LC-MS Chromasolv grade acetonitrile (99.98% pure) was from Riedelde H~ien-Sigma-AldrichChemie (Schnelldorf,Germany). Purified water was obtained in the laboratory using a Milli-Q water system (Le Mont-sur-Lausanne, Switzerland). Methanol, dichloromethane, 2-propanol, formic acid (99%), boric acid, potassium chloride, sodium hydroxide, sodium acetate, and acetic acid (glacial) 100% anhydre were from Merck (Darmstadt, Germany). Ammonium formate and 13-glucuronidase, Type L-II, from Limpets, were from FlukaSigma-Aldrich Chemie (Steinheim, Switzerland). Solid-phase extraction (SPE) cartridges OASIS HLB (3 cc, 60 rag) were from Waters. A pH 3.0 ammonium formate 2mM buffer was prepared by mixing 1 mL ammonium formate, 1M solution (dissolving ammonium formate 3.153 g in 50 mL of water), and 0.5 mL formic acid, and adding water until 500 mL. A pH 9.0 borate buffer was prepared by mixing 6.2 g of boric acid and 7.5 g of potassium chloride with 420 mL of a solution of 0.1 N sodium hydroxide, and adding water until 1000 mL. Fresh drug-free human urines, which were confirmed negative using the described method, were obtained from healthy volunteers.
574
Extraction One milliliter volume of each urine sample was centrifuged for 5 rain at 12,100 xg. To 0.5 mL of the supernatant, 50 IJL of a mixed working solution of IS (morphine-d3, 6-acetylmorphine-d3, codeine-d3,amphetamine-ds, d,l-methamphetaminads, d,I-MDA-ds, d,I-MDMA-ds, ecgonine methyl ester-d3, benzoylecgonine-d3, EDDP-d3, methadone-d3at 1 rag/L, and LSD-d3at 0.1 mg/L in methanol), and I mL of pH 9.0 borate buffer were added. The calibrating standards of urine were prepared by spiking blank urine samples with the appropriate working solution volumes. After conditioning with 2 mL methanol and 2 mL water, the previously prepared samples were applied onto the SPE cartridges. Clean-up was accomplished with successive 2 mL washes of water-methanol (95:5, v/v) and a mixture of water-2%NH~OH in methanol (80:20, v/v). The cartridges were dried for 5 min before elution with 2.5 rnL of a mixture of dichloromethane-2-propanol (70:30, v/v). The elution solution was evaporated to dryness at 40~ under a fine stream of nitrogen. The dry extract was re-dissolvedin 100 IJLof a pH 3.0 ammonium formate buffer. The sample was transferred into autosampler vials, and 20 IJL were injected into the LC-MS-MS. During the whole extraction procedure, the samples were protected from the light. Urine hydrolysis In order to quantify the total morphine and codeine, a urine hydrolysis was performed. To 0.5 mL of urine samples, 0.5 mL of acetate pH 4.5 buffer and 200 IJL of I~-glucuronidaseat 50000 U/L were added. The samples were incubated for 16 h at 40~ in the presence of the IS. Then the samples were kept at the laboratory temperature for 1 h, and 2 mL of borate pH 9 buffer were added. These samples were extracted as described previously. This urine hydrolysis is used as a routine procedure in our laboratory, following the recommendations of Combie et al. (19).
Chromatography Chromatographic separation was performed with a Waters Atlantis dC18, 3 IJm (100 x 2.1 mm i.d.) reversed-phase column at 26~ The mobile phase, delivered at a flow rate of 0.2 mL/min, was a gradient of acetonitrile and a pH 3.0 ammonium formate buffer, programmed as follows: 0% acetonitrile for I min, linearly increased to 50% in 10 min, then held for 2.5 rain, decreased to 0% (original conditions) in 0.5 min, and equilibrated for 5 min, which resulted in a total run time of 18 min. In order to prevent source contamination, the first part of the eluate (until 1 min) and the last part of it (from 14 to 18 min) were sent to waste through a divert valve. MS-MS Best results were obtained with a capillary voltage of 3 kV, source block temperature of 130~ desolvatation gas (nitrogen) heated to 400~ and deliveredat 500 L/h, and cone gas at 50 L/h. Collision cell pressure was 3 x 10-6 Bar of argon. Data were recorded in the MRM mode.A post-column infusion system was used to optimize ionization for all target compounds and internal standards. The mobile phase (pH 3.0 am-
Journal of Analytical Toxicology, Vol. 31, November/December 2007
tee, using a syringe pump at 10 pL/min. The precursor ions, product ions, cone voltage, and collision energy obtained using the autotune wizard are presented in Table I. The MS method was divided into four MRMfunctions to obtain at least 15 acquisition points per peak. Table I. LC(ESI)-MS-MS Parameters, Retention Time, and Corresponding IS Validation Cone Collision The analytical validation was performed acCompound Function Transition voltage(V) energy(eV) RT IS cording to the recommendations of international organizations FDA (20) and ICH (21), EME Function 1: 200.2> 81.8 35 25 2.5 EME-d3 Shah et al. (22), and Peters and Maurer (23). MRM of 200.2 > 182.2' 35 17 The specificity of the method was evaluated EME-d3 3 pair of 203.2 > 185.2 30 18 2.5 by analyzing urine from 15 healthy non-drugmasses consuming subjects. Morphine Function2: 286 > 201.2' 40 25 7 Morphine-d3 The linearity was obtained with an average MRM of 286 > 229.2 40 25 determination coefficient (r2) > 0.99 over a 3 pair of range from the lower limit of quantitation Morphine-d3 masses 289.1 > 201.1 40 25 7 (LLOQ) up to the upper limit of quantitation Codeine Function3: 300.1> 215.2' 40 25 8.1 Codeine-d3 (ULOQ). A weighting factor 1/• was used. MRM of 300.1 > 243.2 40 25 Within-day precision and accuracy were de18 pair of 303.1 >215.1 45 25 8.1 terminated at four concentration levels (the Codeine-d3 masses LLOQ, the ULOQ, and two intermediate A 136 > 90.6* 20 15 8.2 A-ds levels) by preparing and analyzing six repli136 > 119 20 9 cates on the same day, using urine samples from six different sources for each level. BeA-d s 141.2 > 124.1 15 9 8.2 tween-day precision and accuracy were asMDA 180> 104.8 18 20 8.5 MDA-ds sessed by analyzing on six different days, urine 180>163' 18 10 samples from six different sources spiked at MDA-ds 185.1 > 168.1 15 9 8.5 the concentrations of the calibration range. Precision, expressed as the coefficient of vari6-AM 328 > 165.1' 45 40 8.5 MAM-d3 ation (CV) of the measured values, was ex328 > 211.1 45 25 pected to be less than 15% for all 6-AM-d3 331.1 > 165.1 40 45 8.5 concentration levels, except for the LLOQ, for which 20% was acceptable. In the same way, MA 150 > 90.6* 24 17 8.5 MA-ds accuracy was evaluated using the mean rela150> 119 24 11 tive error (MRE), which is the percentage deMA-ds 155.1 > 120.9 22 11 8.5 viation from the accepted reference value, MDMA 194 > 104.8 20 25 8.7 MDMA-ds which had to be less than 15% for all concen194 > 163' 20 12 tration levels, except for the LLOQ,for which 20% was acceptable. MDMA-ds 199.1 > 165 22 12 8.7 The lower limit of detection (LOD) was deBE Function4: 290.1> 104.9 30 30 9.4 BE-d3 fined as the lowest concentration of the drug MRM of 290.1 > 168.2' 30 18 resulting in a signal-to-noise ratio of 3:1. The 12 pair of LLOQ was defined as the lowest concentraBE-d3 masses 293.2 > 171.2 30 20 9.4 tion yielding within-day and between-day CV LSD 324.1 > 223.2* 35 25 10.4 LSD-d3 and MRE less than 20%. 324.1 > 281.2 35 20 The recovery was determined in triplicate at an intermediate concentration level for each LSD-d3 327.2 > 226.3 35 25 10.4 compound. Three blank urines were spiked EDDP 278.3 > 234.4* 45 33 11.9 EDDP-d3 with the appropriate amount of each com278.3 > 249.3 45 25 pound. These fortified samples and three EDDP-d3 281.3 > 234.4 47 31 11.9 blank samples were extracted as previously described. The dry extracts of the fortified Methadone 310.2 > 104.9 20 25 12.4 Methadone-d3 samples were re-dissolved in 100 IJL of the 310.2 > 265.3* 20 15 reconstitution solvent containing the IS, Methadone-d3 313.3 > 268.4 31 15 12.4 while the extracts of the blank samples were re-dissolved in 100 IJL of the reconstitution * The quantitation transition. solvent containing the respective nominal
monium formate buffer-acetonitrile, 50/50) was delivered into the electmspray interface at a rate of 0.2 mL/min while analyte at 10 mg/L was infused post-column through a dead volume
575
Journal of Analytical Toxicology, Vol. 31, November/December 2007
amounts of the compounds and the IS. The latter were used as neat standards. The ion suppression effect on the ESI response was evaluated according to the following procedure (24): ten drug-free urine samples from different sources were extracted as described previously. The extracts were then fortified with all drugs at an intermediate concentration level. Three reference solutions in pH 3 ammonium formate buffer were also fortified with all drugs at the same nominal concentration. The reconstituted extracts and the reference solutions were injected into the LC-MS-MS system. Peak areas obtained from the extracts were compared with the corresponding peak areas produced by the reference solutions. The ion suppression percentage was calculated as follows: (mean peak area "reconstituted extract in urine"- mean peak area "reference solution") • 100 / mean peak area "reference solution". The ion ratio was calculated as the peak area of the quantitation transition / the peak area of the qualifier transition. The relative ion intensity was defined as percentage of the peak area of the quantifier transition (100 / ion ratio), in such a way that, for example, a relative ion intensity of 50% meant that the peak area of the qualifier transition was half peak area of the quantitation transition. Its within-day and between-day variation (CV)was studied. The freeze-and-thaw stability of the analytes was determined after three freeze-and-thaw cycles of fortified urine samples at two concentration levels (10 and 100 pg/L for all compounds,
and 1 and 10 pg/L for LSD). All the concentrations above the ULOQwere diluted with drug-free urine for quantitation. The described method was applied to 25 real samples which had tested positive for any of the studied compounds in a previous enzymoimmunoassay.
Quality control samples The developed method was applied to 24 quality control samples from the "Quality control programme of drugs of abuse analysis in urine samples", conducted by Institut Municipal d'Investigaci6 M~dica, Barcelona, Spain. The percent deviations to the reported nominal value were calculated as follows: (Measured value-Nominal value) • 100 / Nominal value. A regression analysis was also done.
Resultsand Discussion
The ion chromatograms of the 12 drugs of abuse are shown in Figure 1. Under the chromatographic conditions used, there was no interference with any extractable endogenous compounds of urine samples. The linearity of the compound-to-IS peak ratio versus the theoretical concentration was verified in urine by using a 1/x weighted linear regression. The determination coefficients were > 0.99 and the curvature was tested on a set of six calibration curves. The calibration range, with at least 9 levels, was 1-2000 pg/L for ecgonine methyl ester, benzoylecgonine, 6-acetylmorphine, methamphetamine, MDMA,and EDDP; 2-2000 pg/L for morphine, codeine, MDA,and methadone; 2-1000 pg/L for amphetamine; ~00 ig "r-''i'0o'" 10:00 i'~ 1200 13~ and 0.2-100 pg/L for LSD. This wide range allows analyzing and quantifying in the same ~ - ~ " ........ ~ , ~ " " - - ; ~ : ~ o ' - - ' - - ~ B~ ' -~o~ ~o'| ,,0o ~z:| ,30o batch urine samples with low concentrations ~ rlTz~vi~o~loc~ine ,3'~:: (like DFSA cases) and urine samples with high ~ 2~ ~0r 4oo ~0o 60o v0o ~0o g0o iooo 110o 1200 1500 concentrations, not requiring a previous dilution step. The within-day and between-day precision mO~ M~hlmphetarnirle ;'~ :~~-I and accuracy were satisfactory for all tested o] ;/ concentrations. Recovery was > 65% for all compounds. Matrix effect ranged, in absolute 300 I0o Io0o 11oo 12"oo ~30o value, from 6 to 43%, depending on the drug. These results are shown in Table II. During .............................................................. ~.;i!,!...._~ e~ ~'~ 80o 1~0o the development of the present method, matrix effect was a difficult challenge to handle. ~| ~| 400 s| ~| ~ ' ~ " , ~ . . . . . , o ~ . . . . . . .,.~. . . . . . . . . . . .~=~ ,~| ' Different SPE cartridges, wash steps, and chromatographic conditions were assayed, but the best results for avoiding matrix effect were ob:~ .... tained by modifying the elution solvent of the solid-phase extraction. In any case, despite the ~oo 30o 400 I~0o ~m ~ ~ ooo Iooo 11oo ~:IOO 13oo fact that matrix effect was observed for certain compounds, no important variations were ;=i ....... 'Z " = found between urines from different sources Time (rain) (CV < 29.3%). Figure1. LC-MS-MSquantitativetransitionchromatogramsof a urinesamplespikedat 5 IJg/l_ The LOQ was the lowest level of the caliof all compounds,exceptLSDat 0.5 pg/L bration curve, and the LOD was 0.1 pg/L for .
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Journalof AnalyticalToxicology,Vol. 31, November/December2007
Table II. Validation Parameters Results
Compound EME
BE
MA
MDA
MDMA
Morphine
Codeine
6-AM
Methadone
EDDP
LSD
Concentration (ng/mL)
Within-day precision and accuracy(n = 6) Mean
Between.dayprecision and accuracy (n -- 6)
CV
MRE
Mean
CV
MRE
1 10 1O0 2000
0.99 10.3 102.41 2019.77
7.91 3.88 2.29 3.09
-1.11 3 2.41 0.99
0.97 10.1 101.4 1999.72
16.89 3.49 6.55 2.55
-3.33 1 1.4 0.01
1 10 100 2000
0.91 10.81 99.81 2016.31
8.58 3.74 1.97 1.68
-8.89 8.11 -0.19 0.82
1 10.15 99.92 1999.65
12.65 5.84 7.78 1.32
0 1.5 0.08 0.02
2 10 100 1000
1.7 10.6 105.38 967.34
11 2.11 5.79 3.98
-15 6 5.38 -3.27
1.67 10.4 107.85 983.22
18.07 5.12 3.85 1.99
-16.67 4 7.85 -1.68
1 10 100 2000
0.9 10.67 104.56 2056.07
5.56 3.81 3.04 2.71
-10 6.67 4.56 2.8
0.98 10.32 104.98 1994.02
11.89 4.18 5.21 2.72
-1.67 3.17 4.98 -0.3
2 10 100 2000
1.87 10.68 97.81 2026.5
7.09 5.46 4.07 1.6
-6.67 6.78 -2.19 1.33
1.9 10.33 102.78 1993.67
13.72 1.58 4.68 1.58
-5 3.33 2.78 -0.32
1 10 100 2000
0.99 10.61 102.26 2046
3.37 4.12 2.12 2.43
-1.11 6.11 2.26 2.3
1.03 10.05 101.45 2002.3
11.72 4.07 5.77 0.82
3.33 0.5 1.45 0.11
2 10 100 2000
2.22 10.79 101.19 1984.89
5.86 4.28 2.53 1.29
11.11 7.89 1.19 -0.76
2.02 10.28 101.4 2003.58
11.49 2.08 4.61 0.9
0.83 2.83 1.4 0.18
2 10 100 2000
2.23 10.89 97.01 2016.28
8.38 6.77 5.1 2.2
11.67 8.89 -2.99 0.81
2.13 10.4 100.73 2003.45
11.35 4.87 7.03 2.23
6.67 4 0.73 0.17
1 10 100 2000
0.98 9.88 98 2007.33
13.31 4.23 5.67 2.88
-2.22 -1.22 -2 0.37
1.05 10.42 101.37 2000.52
14.44 4.44 4.11 18
5 4.17 1.37 0.03
10.63 104.56 1986.17 2.3
2.04 1.44 6.33 4.56
-0.69 10.52 103.12 2000.28
14.13 7.87 4.15 1.12
-3.33 5.17 3.12 0.01
2 10 100 2000 1 10 1O0 2000 0.2 1 10 100
2.21 2.72 10.56 1.93 0.87 10.4 99.99 2052.98 0.2 1.1 10.04 102.14
5.77 2.88 2.61 1.73
-13.33 4 -0.01 2.65
1.02 10.3 101.28 2008.38
11.5 4.03 4.08 1.1
1.67 3 1.28 0.42
0 7.87 2.69 2.32
0 10 0.44 2.14
0.2 1.05 10.13 102.03
0 5 4.61 1.18
0 3.33 1.33 2.03
Matrix Recovery
effect (n = 10)
(%)
%
CV
65.78
19
3.4
77.3
-43
23.7
86.58
-16
19.1
90.23
-9
24.8
80.78
-12
8.8
83.98
-30
25.8
72.51
-17
20.8
82.7
--23
29.3
82.72
-31
16.4
80.15
15
17.6
75.65
86
20.5
-35
27.3
577
Journal of Analytical Toxicology, Vol. 31, November/December 2007
Table III. Relative Ion Intensity Data Between-dayrelative Between-dayrelative ion intensities(%) ion intensities(%) at low concentration at highconcentration
Within-day relative ion intensities(%) Compound
Mean
CV
Mean
CV
Mean
CV
EME BEG A MA MDA MDMA Morphine Codeine 6-AM Methadone EDDP LSD
55.6 27.8 58.8 66.7 21.7 26.3 38.5 37 62.5 50 50 22.2
3.7 5.8 9.8 2.7 5.8 2.2 4.7 6.8 6.3 3.3 6.3 5.6
66.7 32.3 50 55.6 29.4 32.3 40 40 66.7 55.6 52.6 22.2
33.1 32 22.9 21.7 34 37.3 11.5 14.8 17.4 18.5 7.4 14.5
62.5 32.3 58.8 58.8 27 33.3 38.5 38.5 71.4 52.6 52.6 20.8
33 31.3 23.8 20.9 38.9 37.7 4 5.4 17.5 19.7 6.1 10.1
Table IV. Freeze-and-Thaw Stability at Low and High Concentration Levels (10 and 100 pg/L for all compounds, and 1 and 10 pg/L for LSD) Stabilityat low concentration(n = 3)
Stabilityat low concentration(n = 3)
Compound
Mean
CV
MRE
Mean
CV
MRE
EME BEG A MA MDA MDMA Morphine Codeine 6-AM Methadone EDDP LSD
9,2 10.5 9.7 10.6 10.8 10,1 10,6 10,5 10,9 10 10.7 1.1
2,2 2.5 2.5 2.5 3.6 2.2 1,9 3.3 5.5 2 2.8 3.4
-7.9 4,7 -3.4 6.2 7.9 0.5 6.1 4.5 8.6 0.2 6,6 7
88.8 97.5 98.9 102,8 102,9 98,2 99 97,4 100.4 94 98.5 9.8
5.2 2,1 2.4 4.4 5.2 2.3 2.4 2.6 5,3 2.2 1.1 4.4
-11.2 -2.5 -1.1 2.8 2.9 -1.8 -1 -2,6 0.4 -6 -1.5 -1.8
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LSD; 0.5 pg/L for ecgonine methyl ester, benzoylecgonine, methamphetamine, MDMA, 6monoacetylmorphine and EDDP; and 1 pg/L for amphetamine, MDA, morphine, codeine, and methadone. In most of the cases, the relative ion intensities, calculated as previously mentioned at each concentration level, showed a within-day (n = 6) CV r 10% but a between-day (n = 6) CV > 20%. It was observed that the within-day relative ion intensities were constant (CV r 10%) through all the concentration levels for each compound. Table III shows the mean and CV calculated withinday and between-day.The within-day data were calculated with the relative ion intensities obtained on the same day at all the concentration levels,and the between-day data were calculated with the relative ion intensities obtained on six different days at low concentration (0.5 pg/L for LSD and 5 pg/L for the other compounds) and high concentration (50 pg/L for LSD and 500 pg/L for the other compounds). Maralikova et al. (16) showed the relative ion intensities for morphine, cocaine, methadone, THC, their metabolites, and LSD by analyzing six different plasma samples spiked at 5 and 50 pg/L, performed on two separate days. These results fulfilled criteria for permitted variations of relative intensities of MRM transitions (8,9). In the case of our method, we observed that the criteria were satisfied within-day for all compounds, and betweenday for methamphetamine, morphine, codeine, 6-acetylmorphine, methadone, EDDP, and LSD. According to this, relative ion intensity should be taken only with caution as an identification criterion. Due to the low within-day variations and the high betweenday variations for some compounds, it is strongly recommended to analyze spiked samples and the real cases in the same day, because within-day relative ion intensity variations satisfied the established criteria
(8,9).
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Figure2. LC-MS-MS quantitative and qualitative transition chromatograms of a real case positive for ecgonine methyl ester (244.7 lag/L)and benzoylecgonine (758.2 i.Jg/L).
578
The present method also allows the determination of MDEA, MBDB, and 2-oxo-3-hydroxy-LSD, but their validation was not performed. The sample volume employed in this method is a bit high for a LC-MS-MS method, but it was necessary for achieving 0.2 pg/L as LSD LOQ. Analyte recoveries in the stability experiments were within the permitted variability range. No significant loss or deterioration for any of the compounds of interest was observed. These results are shown in Table IV.
Journalof AnalyticalToxicology,Vol. 31, November/December2007
Conclusion An LC-MS--MSmethod was developedand fully validated for the simultaneous determination of 12 compounds (cocaine metabolites, amphetamines, opiates, methadone, and LSD), taking into account the criteria for confirmation of compound identity. The wide calibration range (1-2000 pg/L, 0.2-100 pg/L) made it a useful confirmation method for both routine analysis and DFSAcases.
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This work was done thanks to the financial support from the Ministerio de Educaci6n y Ciencia of Spain (F.P.U. Grant number AP-2002-2935 and AP-2002-2878).
Figure 3. Regressionanalysisof the quality control results (y = 1.0177x, r2 = 0.9336).
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Twenty-five real cases were analyzed using this method. Twenty-two were confirmed positive for cocaine metabolites, 2 for opiates, and I for methadone and EDDP.Figure 2 shows a chromatogram from a real case. The method was also applied to 24 quality control samples from the "Quality control programme of drugs of abuse analysis in urine samples", conducted by Institut Municipal d'Investigaci6 M~dica, Barcelona, Spain.All the quality control samples were correctly identified as positive or negative for each compound; 3 urine samples tested positive for amphetamine, 1 for methamphetamine, 2 for MDA,2 for MDMA, 5 for morphine, 4 for codeine, 6 for benzoylecgonine, 2 for ecgonine methyl ester, 2 for LSD, 2 for EDDP, and 3 for methadone. The percent deviations from the reported nominal value ranged from 1.7 to 37%. All compounds showed a deviation < 25%, except metharnphetamine (37%). Figure 3 shows the regression analysis obtained, y = 1.0177x, r2 = 0.9336, which is satisfying. A chromatogram from quality control urine is shown in Figure 4.
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