Development and Validation of a High-Performance Liquid

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of amphetamine, methamphetamine, and ...
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Anal. Chem. 2004, 76, 2124-2132

Development and Validation of a High-Performance Liquid Chromatography-Mass Spectrometry Assay for Determination of Amphetamine, Methamphetamine, and Methylenedioxy Derivatives in Meconium Simona Pichini,*,†,‡ Roberta Pacifici,† Manuela Pellegrini,† Emilia Marchei,† Jaime Lozano,‡,§ Janeth Murillo,‡,§ Oriol Vall,§ and O Ä scar Garcı´a-Algar§

Drug Research and Control Department, Istituto Superiore di Sanita` , V. le Regina Elena 299, 00161 Rome, Italy, Universitat Auto` noma, Barcelona, Spain, and Paediatric Service, Hospital del Mar, Passeig Maritim, 08003 Barcelona, Spain

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of amphetamine, methamphetamine, and methylendioxy derivatives in meconium, using 3,4-methylendioxypropylamphetamine as internal standard. The analytes were initially extracted from the matrix by 17 mM methanolic HCl. Subsequently, a solid-phase extraction with Bondelut Certify columns was applied. Chromatography was performed on a C18 reversed-phase column using a linear gradient of 10 mM ammonium bicarbonate, pH 9.0methanol as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with an atmospheric pressure ionization-electrospray interface. The method was validated in the range 0.005-1.00 µg/g using 1 g of meconium per assay. Mean recoveries ranged between 61.1 and 87.2% for different analytes. The quantification limits were 0.005 µg/g meconium for amphetamine, methamphetamine, and 4-hydroxy-3-methoxymethamphetamine and 0.004 µg/g meconium for 3,4methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine. The method was applied to analysis of meconium in newborns to assess eventual fetal exposure to amphetamine derivatives.

Drug abuse during pregnancy is a major problem because of the associated high incidence of perinatal complications and high morbidity and mortality rates of newborns. Various neonatal birth defects are thought to be related to fetal exposure to drugs, chemical agents, or other xenobiotics.1 * To whom correspondence should be addressed. Fax ++39 06 49902016. E-mail: [email protected]. † Istituto Superiore di Sanita`. ‡ Universitat Auto`noma. § Hospital del Mar. (1) Moore, C.; Negrusz, A.; Lewis, D. J. Chromatogr., B 1998, 713, 137-146.

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During the past decades, urine has been the specimen of choice for drugs of abuse screening also at delivery.2 However, drugs present in the urine reflect consumption or exposure during the preceding one to four days.3 Investigators have reported the utility of meconium as a test specimen in the screening of newborns for drug abuse.4-6 Meconium is the first fecal matter passed by a neonate. Its formation starts between the 12th and 16th week of gestation and usually accumulates in fetal bowel until birth, and it is passed by the neonate one to five days after birth. For this reason, meconium analysis extends the window of detection of drug use to approximately the last 20 weeks of gestation, being more informative than urine for the detection of drug exposure in pregnancy.7-8 We recently developed methodologies for the determination of opiates, cocaine, and arecoline (areca nut alkaloid) in meconium and investigated the presence of these drugs in samples from newborns enrolled in a pilot study aimed to estimate chronic fetal exposure to pharmaceuticals, drugs of abuse, and tobacco smoke in Italy and Spain (The Meconium Project).9-10 Preliminary results disclosed that 6 and 8.5% of newborns tested positive for opiates and cocaine, respectively, and 2 out of 32 Asiatic newborns tested positive for arecoline.11 (2) Moriya, F.; Chan, K.; Noguchi, T. T.; Wu, P. Y. K. J. Anal. Toxicol. 1994, 18, 41-45. (3) Huestis, M. A.; Choo, R. E. Forensic Sci. Int. 2002, 128, 20-30. (4) Ostrea, E. M.; Parks, P.; Brady, M. Clin. Chem. 1988, 34, 2372-2373. (5) Lewis, D.; Moore, C.; Leikin, J. B.; Kechavarz, L. Vet. Hum. Toxicol. 1995, 37, 318-319. (6) Pichini, S.; Altieri, I.; Zuccaro, P.; Pacifici, R. Clin. Pharmacokinet. 1996, 31, 81. (7) Koren, G.; Chan, D.; Klein, J.; Karaskov, T. Ther. Drug Monit. 2002, 24, 23-25. (8) Bar-Oz, B.; Klein, J.; Karaskov, T.; Koren, G. Arch. Dis. Child Fetal Neonatal Ed. 2003, 88, F98-F100. (9) Pichini, S.; Pacifici, R.; Pellegrini, M.; Marchei, E.; Pe´rez-Alarco´n, E.; Puig, C.; Vall, O.; Garcı´a-Algar, O. J. Chromatgr., B 2003, 794, 281-292. (10) Pichini, S.; Pellegrini, M.; Pacifici, R.; Marchei, E.; Murillo, J.; Puig, C.; Vall, O.; Garcı´a-Algar, O. Rapid Commun. Mass Spectrom. 2003, 17, 19581964. (11) Pichini, S.; Zuccaro, P.; Marchei, E.; Pellegrini, M.; Pe´rez-Alarco´n, E.; Puig, C.; Vall, O.; Pacifici, R.; Ordobas, L.; Garcı´a-Algar, O. In European Collaboration: towards drug development and rational drug therapy; Tulunay, F. C., Orme, M., Eds.; Springer: Berlin 2003; p 105. 10.1021/ac035419x CCC: $27.50

© 2004 American Chemical Society Published on Web 02/24/2004

On the basis of findings in humans and the confirmation of prenatal exposure in animals, it is known that amphetamine (AP) and methamphetamine (MA) increase the risk of an adverse outcome when abused during pregnancy.12 Malformations and adverse outcomes reported for the use of amphetamine or methamphetamine during pregnancy include cleft lip, cardiac defects, biliary atresia, cerebral hemorrahage, low birth weight, growth reduction, reduced head circumference, and altered neonatal behavioral patterns.13-14 Conversely, information regarding the consequences of fetal exposure to methylenedioxy derivatives of amphetamine (e.g., 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA)) is limited to animal models.15-16 To investigate eventual fetal exposure to amphetamine derivatives in our study population, the development of a more easily used, sensitive, and specific method for determination of different analytes in meconium was found to be necessary. A large number of methods for the determination of AP, MA, and related compounds in biological samples have been reported using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS).17-20 GC/ MS is widely accepted as the definitive analytical method, because of its sensitivity and selectivity and the ease of identification of compounds from mass spectra. However, GC and GC/MS generally require derivatization prior to analysis in order to improve their chromatographic properties. More recently, LC-MS has increased popularity due to the fact that it does not require sample derivatization, allows simultaneous determination of lipophilic parent drugs and hydrophilic metabolites, and employs a simplified sample preparation.21 The present paper describes a sensitive and selective analytical method based on HPLC-MS using electrospray ionization detection for the determination of amphetamine, methamphetamine, and methylenedioxy derivatives in meconium using 3,4-methylenedioxypropylamphetamine (MDPA) as the internal standard. A solid-phase extraction method coupled with methanolic treatment of meconium specimens was used to simultaneously extract AP, MA, MDMA, and its metabolites MDA and 4-hydroxy-3methoxymethamphetamine (HMMA), 3,4-methylenedioxyethylamphetamine (MDEA), N-methyl-1-(3,4-methylenedioxyphenyl)2-butanamine (MBDB), and MDPA. EXPERIMENTAL SECTION Chemicals. Standards of AP, MA, MDA, MDMA, and MDPA were supplied by Salars (Como, Italy). HMMA and MDEA with (12) Plessinger, M. A. Obstet. Gynecol. Clin. North Am. 1998, 25, 119-138. (13) Oro, A. S.; Dixon S. D. J. Pediatr. 1987, 111, 571-578. (14) Little, B. B.; Snell, L. M.; Gilstrap, L. C. Obstet. Gynecol. 1988, 72, 541544. (15) Bronson, M. E.; Jiang, W.; Clark, C. R.; De Ruiter J. Brain Res. Bull. 1994, 34, 143-150. (16) Koprich, J. B.; Chen, E. Y.; Kanaan, N. M.; Campbell, N. G.; Kordower, J. H.; Lipton, J. W. Neurotoxicol. Teratol. 2003, 25, 509-517. (17) Pizarro, N.; Ortun ˜o, J.; Farre´, M.; Hernandez-Lopez, C.; Pujadas, M.; Llebaria, A.; Joglar, J.; Roset, P. N.; Mas, M.; Segura, J.; Cami, J.; de la Torre R. J. Anal. Toxicol. 2002, 26, 157-165. (18) Pujadas, M.; Pichini, S.; Poudevida, S.; Menoyo, E.; Zuccaro, P.; Farre´, M.; de la Torre R. J. Chromatgr., B, in press. (19) Kraemer, T.; Maurer, H. H. J. Chromatgr., B 1998, 713, 163-187. (20) Maurer, H. H. J. Chromatgr.. B 1998, 713, 3-25. (21) Kataoka, H.; Lord, H. L.; Pawliszyn, J. J. Anal. Toxicol. 2000, 24, 257265.

Table 1. Mobile-Phase Gradient Table in HPLC Separation A (%), ammonium bicarbonate, pH 9.0

B (%), methanol

start time (min)

70 70 50 50 70 70

30 30 50 50 30 30

0 11 12 18 19 30

MBDB were kindly donated by Dr. Rafael de la Torre (Institut Municipal d’Investigacio´ Me`dica, Barcelona, Spain) and Prof. Annunziata Lopez (Istituto di Medicina Legale e delle Assicurazioni, Universita` La Sapienza, Rome, Italy), respectively. Bond Elut Certify solid-phase extraction (SPE) columns were from Varian (Palo Alto, CA). Ultrapure water and all other reagents of analytical grade were obtained from Carlo Erba (Milan, Italy). Meconium Samples. Meconium samples came from the Hospital de Mar of Barcelona, Spain (the fourth largest hospital in the city) as part of the “Meconium Project” joint Italian-Spanish study. The study protocol, which was approved by the local ethical committee (CEIC-IMAS, Barcelona, Spain), started at the beginning of 2002, and meconium specimens from at least 1000 newborns were expected to be collected and analyzed to assess the prevalence of fetal exposure to drugs. Up to now, 700 samples have been collected, aliquoted, and stored at -20 °C until analysis. Additional meconium samples were kindly donated by Dr. Christine Moore (U.S. Drug Testing Laboratories, Des Plaines, IL). Instrumentation. LC-MS analyses were performed using an Agilent 1100 series HPLC system consisting of a G1312A binary pump, a G1322A degasser, and an ALS G1329A autosampler (Agilent Technologies, Palo Alto, CA) interfaced to an Agilent 1100 series G1946D mass spectrometer equipped with an atmospheric pressure ionization-electrospray (ESI) interface. Chromatographic separation was achieved using a Waters Xterra RP 18 column (150 × 2.1 mm; 5 µm) (Waters, Rome, Italy). The mobile phase used in the separation consisted of (A) 10 mM ammonium bicarbonate, pH 9.0, and (B) methanol. The gradient program is shown in Table 1. The flow rate was 0.3 mL/min. All chromatographic solvents were degassed with helium before use. The injection volume was 20 µL, and the column temperature was set at 40 °C. The mass spectrometer was operated in positive ESI mode with selected ion monitoring (SIM) acquisition. The following ESI conditions were applied: drying gas (nitrogen) heated at 350 °C at a flow rate of 12.0 L/min; nebulizer gas (nitrogen) at a pressure of 50 psi; capillary voltage at 4000 V; fragmentor voltage (applied to the exit end of the capillary) at 110 V; dwell time at 139 ms; and mass peak width at 0.10 min. Qualifying ions were m/z 136, 119, and 91 for AP; m/z 150, 119. and 91 for MA; m/z 180, 163. and 105 for MDA; m/z 194, 163, and 105 for MDMA; m/z 196, 165, and 105 for HMMA; m/z 208, 163, and 105 for MDEA; m/z 208, 177, and 135 for MBDB; and m/z 222, 163, and105 for MDPA. The acceptance criterion for ion intensity ratios was a deviation of e20% of the average of the ion intensity ratios of all the calibrators. The [M + H]+ ions Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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at m/z 136 for AP, m/z 150 for MA, m/z 180 for MDA, m/z 194 for MDMA, m/z 196 for HMMA, m/z 208 for MDEA, m/z 208 for MBDB, and m/z 222 for MDPA were selected for quantification. Calibration Standards and Quality Control Samples. Stock standard solutions (1 mg/mL) of analytes were prepared in methanol. Working solutions at concentrations of 10, 1, and 0.1 µg/mL were prepared by dilution of the stock standards with methanol and stored at -20 °C until analysis. The internal standard (IS) MDPA working solution was used at a concentration of 10 µg/mL. Calibration standards containing 1, 0.5, 0.1, 0.05, 0.01, and 0.005 µg/g of meconium were prepared daily for each analytical batch by adding suitable amounts of methanol working solutions to 1 g of prechecked drug-free meconium pool. Quality control samples of 0.85 (high control), 0.12 (medium control), and 0.012 µg/g (low control) and samples at the limit of quantification (LOQ) of each analyte were prepared in drug-free meconium and stored at -20 °C. They were included in each analytical batch to check calibration, accuracy, precision, and the stability of samples under storage conditions. Sample Preparation. A 1-g amount of meconium with 10 µL of IS working solution was transferred into 15-mL screw-capped glass tubes, and 3 mL of methanol-HCl was added. The tubes were placed in a horizontal shaker for 20 min. After centrifugation at 2000 rpm for 10 min, the organic layer was transferred to another tube and the solvent was evaporated to dryness at 30 °C under a nitrogen stream. The residue was dissolved in 2 mL of 0.1 M phosphate buffer, pH 6.0, and applied on a Bond Elut Certify SPE column, which had been preconditioned with 2 mL of methanol and 2 mL of 0.1 M phosphate buffer, pH 6.0. The column was washed with 1 mL of 1.0 M acetic acid and 4 mL of methanol. The analytes were eluted with 2 mL of ethyl acetate with 2% ammonium hydroxide. The eluent was evaporated to dryness under a stream of nitrogen and redissolved in 100 µL of 10 mM ammonium bicarbonate, pH 9.0. A 20-µL volume was injected into the HPLC column. Validation Procedures. Prior to application to real samples, the method was tested in a 3-day validation protocol following the accepted criteria for bioanalytical method validation.21,22 Selectivity, recovery, matrix effect, linearity, precision, accuracy, and limits of detection and quantification were assayed. A total of 20 different meconium samples from newborns, whose mothers had a negative history of illicit drug exposure during pregnancy, were extracted and analyzed for assessment of potential interferences due to endogenous substances. The apparent responses at the retention times of the analytes under investigation and IS were compared to the response of analytes at the LOQ and IS at its lowest quantifiable concentration. Furthermore, potential interferences from principal drugs of abuse, opiates (6-monoacetylmorphine, morphine, morphine 3-glucuronide, morphine 6-glucuronide, codeine), cocaine and metabolites (benzoylecgonine and cocaethylene), cannabinoids (∆-9-tetrahyrocannabinol, 11-nor-∆-9-tetrahydrocannabinol-9-carboxylic acid), benzodiazepines (clorazepate, diazepam, lorazepam, oxazepam, alprazolam), and antidepressants (imipramine, clomipramine, (22) Guidance for Industry, Bioanalytical Method validation, U.S. Department of Health and Human Services, Food and Drug Administration, May 2001 (http://www.fda.gov/cder/guidance/4252fnl.htm).

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fluoxetine, norfluoxetine, paroxetine), were also evaluated by spiking 1 g of prechecked drug-free meconium pool with 1 µg of each of the aforementioned substances and carrying out the entire procedure. The potential for carryover was investigated by injecting extracted blank meconium, with added internal standard, immediately after analysis of the highest concentration point of the calibration curve on each of the 3 days of the validation protocol and measuring the area of eventual peaks present at the retention times of analytes under investigation. Analytical recoveries were calculated by comparing the peak areas obtained when calibration samples were analyzed by adding the analytical reference standard and the internal standard in the extract of drug-free meconium prior to and after the extraction procedure. The recoveries were assessed at three concentration levels (1, 0.05, and 0.005 µg/g), using four replicates at each level. For an evaluation of matrix effects, the peak areas of extracted blank samples spiked with standards at three concentration levels (1, 0.05, and 0.005 µg/g) after the extraction procedure were compared to the peak areas of pure diluted substances. Calibration curves were tested over the quantification limit (0.004 and 0.005 µg/g depending on the considered analyte)s1 µg/g range for all the analytes. Peak area ratios between compounds and IS were used for calculations. A weighted (1/concentration) least-squares regression analysis was used (SPSS, version 9.0.2 for Windows). Five replicates of blank meconium samples were used for calculating the limits of detection and quantification. Standard deviation (SD) of the mean noise level over the retention time window of each analyte was used to determine the detection limit (LOD ) 3 SD) and the quantification limit (LOQ ) 10 SD). A total of five replicates at each of three different concentrations of standards (LOQ, 0.12, and 0.85 µg/g) added to drug-free meconium samples, extracted as reported above, were analyzed for the determination of intra-assay precision and accuracy. The interassay precision and accuracy were determined for three independent experimental assays of the aforementioned replicates. Inter-run precision was expressed as the relative SD (RSD) of concentrations calculated for quality control samples. Inter-run accuracy was expressed as the relative error of the calculated concentrations. The effect of three freeze-thaw cycles (storage at -20 °C) on the compound stability in meconium was evaluated by repeated analysis (n ) 3) of quality control samples (0.012, 0.12, and 0.85 µg/g) for all the analytes. The stability was expressed as a percentage of the initial concentration of the analytes spiked in meconium and quantified just after preparation. RESULTS AND DISCUSSION Mass Spectra. Since ESI is a soft ionization technique with little fragmentation of the molecule, the protonated molecular ion of each compound always was the most abundant one. Nonetheless, the fragmentor voltage of 110 V was chosen as the best compromise value to have the protonated molecular ion as the most abundant one and at least two other characteristic fragment ions with a relative abundance higher than 20%. For both AP and MA, these fragment ions were [C6H5CH2CHCH3]+ at m/z 119 and [C6H5CH2]+ at m/z 91, both with relative abundance between 30 and 60%. For MDMA, MDA, MDEA, and MDPA, the fragments [CH2O2C6H3CH2CHCH3]+ at m/z 163 (relative abundance ranging

Figure 1. SIM chromatogram of an extract of 1 g of drug-free meconium sample spiked with 0.05 µg of HMMA, AP, MA, MDA, MDMA, MDEA, and MBDB and MDPA as internal standard.

from 30 to 70% depending on the investigated compound) and [CH2O2C6H3]+ at m/z 105 (relative abundance ranging from 20 to 50% depending on the investigated compound) were observed. This last fragment was present also in the case of HMMA (20% relative abundance), and the 4-hydroxy-3-methoxy group, which

substitutes the methylenedioxy group of the parent drug, gave rise to the other fragment [CH3OC6H3OHCH2CHCH3]+ at m/z 165 (50% relative abundance). Finally, MBDB showed fragments [CH2O2C6H3CH2CHCH2CH3]+ at m/z 177 (20% relative abundance) and [CH2O2C6H3CH2]+ at m/z 135 (50% relative abundance). Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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Figure 2. SIM chromatogram of an extract of 1 g of drug-free meconium sample.

Validation Results. A representative chromatogram obtained following the extraction of 0.05 µg of AP, MA, MDA, MDMA, HMMA, MDEA, and MDBD with MDPA as IS spiked in 1 g of drug-free meconium is shown in Figure 1. Each chromatographic 2128

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run was completed in 30 min. No additional peak due to endogenous substances that could have interfered with the detection of compounds of interest was observed (Figure 2). None of the drugs of abuse or aforementioned medications, carried

Table 2. Method Calibration in Meconium analyte

calibration line slope (n ) 3)

calibration line intercept (n ) 3)

correlation coefficient (r2)

LOD (n ) 5) (µg/g)

LOQ (n ) 5) (µg/g)

HMMA AP MA MDA MDMA MDEA MBDB

4.545 ( 0.14 2.165 ( 0.42 8.332 ( 0.14 3.681 ( 0.17 7.710 ( 0.11 17.479 ( 0.41 3.61 ( 0.63

0.041 ( 0.01 0.014 ( 0.03 0.029 ( 0.01 0.020 ( 0.01 0.018 ( 0.01 0.168 ( 0.05 0.039 ( 0.02

0.995 ( 0.005 0.994 ( 0.004 0.998 ( 0.001 0.998 ( 0.001 0.999 ( 0.001 0.998 ( 0.001 0.994 ( 0.001

0.001 0.001 0.001 0.001 0.001 0.001 0.001

0.005 0.005 0.005 0.004 0.004 0.004 0.004

Table 3. Intraday (n ) 5) and Interday (n ) 15) Precision and Accuracy intraday

interday

analyte

concn

estimated mean ( SD

precision (RSD)

accuracy (error %)

estimated mean ( SD

precision (RSD)

accuracy (error %)

HMMA

0.005 0.12 0.85 0.005 0.12 0.85 0.005 0.12 0.85 0.004 0.12 0.85 0.004 0.12 0.85 0.004 0.12 0.85 0.004 0.12 0.85

0.0045 ( 0.0004 0.106 ( 0.005 0.821 ( 0.081 0.0048 ( 0.0002 0.102 ( 0.005 0.832 ( 0.062 0.0045 ( 0.0004 0.108 ( 0.004 0.833 ( 0.082 0.0037 ( 0.0001 0.107 ( 0.003 0.836 ( 0.054 0.0035 ( 0.0002 0.106 ( 0.005 0.812 ( 0.063 0.0036 ( 0.0002 0.109 ( 0.003 0.831 ( 0.031 0.0034 ( 0.0001 0.107 ( 0.002 0.821 ( 0.052

8.8 4.7 9.7 4.1 4.9 7.2 8.8 3.7 9.6 2.7 2.8 6.0 5.7 4.7 7.4 5.5 2.7 3.6 2.9 1.8 6.0

10.0 11.6 3.5 4.0 15.0 2.3 10.0 10.0 2.3 7.5 10.8 2.3 12.5 11.6 4.7 10.0 9.1 2.3 15.0 10.8 3.5

0.0045 ( 0.0007 0.102 ( 0.002 0.821 ( 0.050 0.0043 ( 0.0006 0.102 ( 0.002 0.814 ( 0.074 0.0043 ( 0.0006 0.104 ( 0.004 0.832 ( 0.061 0.0036 ( 0.0001 0.102 ( 0.007 0.840 ( 0.014 0.0036 ( 0.0002 0.105 ( 0.005 0.812 ( 0.070 0.0034 ( 0.0002 0.109 ( 0.004 0.843 ( 0.028 0.0035 ( 0.0003 0.108 ( 0.002 0.831 ( 0.021

15.0 1.9 6.0 13.9 2.8 9.1 13.3 3.8 7.3 2.7 7.0 1.6 5.5 4.7 8.6 5.8 3.6 3.3 8.5 1.8 2.5

10.0 15.0 3.5 14.0 12.5 4.7 10.0 13.3 2.3 10.0 15.0 1.1 10.0 12.5 4.7 15.0 9.1 1.1 12.5 10.0 2.3

AP MA MDA MDMA MDEA MBDB

through the entire procedure, interfered with the assay. Blank samples injected after the highest point of the calibration curve did not present any traces of carryover. The average recoveries obtained after methanolic and SPE extraction of meconium were around 80 and 90% (with ∼5% standard deviation between different concentration levels) for HMMA, MDA, MDEA, MBDB and MA, and MDMA. respectively. Lower recovery figures were obtained only in for AP, ranging from 60 to 70%. These results suggested that there was no relevant difference in extraction recovery at different concentration levels for the analytes under investigation. With respect to the matrix effect, the comparison between peak areas of analytes spiked in extracted blank meconium samples versus those for pure diluted standards showed less than 10% analytical signal suppression due to coeluting endogenous substances. Linear calibration curves were obtained for the compounds of interest with a correlation coefficient (r2) higher than 0.99 in all cases (Table 2); limits of detection and quantification were considered adequate for the purposes of the present study (Table 2). Table 3 shows the results obtained for intra-assay and interassay precision and accuracy calculations for all the amphetamine derivatives. These results satisfactorily met the internationally established acceptance criteria.22,23

With reference to the freeze-thaw stability assays for quality control samples, no relevant degradation was observed after any of the three freeze-thaw cycles, with differences from the initial concentration lower that 10%. Analysis of Meconium Samples. The method presented here was used to assess fetal exposure to amphetamines in meconium samples collected at the Hospital del Mar in Barcelona, Spain, and enrolled in the Meconium Project. Up to 600 samples have been analyzed to date with none positive for AP or MA and only one providing positive results for MDMA alone at very low concentration without the presence of HMMA or MDA (Figure 3 and Table 4). This particular sample came from the only mother of our cohort who declared sporadic consumption of “amphetamines” together with opiates and cocaine. This low prevalence of amphetamine derivatives consumption during pregnancy in our cohort is in agreement with previous studies in the North American population.5 In case of AP and MA, it can be explained with the evidence that these two compounds are seldom abused in the Mediterranean areas,24 while for MDMA, the hypothesis is that since this drug is used recreationally, it is (23) ICH Topic Q 2 B Validation of Analytical Procedures: Methodology, The European Agency for the Evaluation of Medicinal Products (http://www.emea.eu.int/htms/human/ich/quality/ichfin.htm). ICH Technical Coordination: London, November 1996.

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Figure 3. SIM chromatogram of an extract of sample 569 containing 0.012 µg/g MDMA.

probably discontinued once pregnancy is known.25 In any case, to verify the feasibility of the developed methodology, additional samples from the United States donated by Dr. Christine Moore 2130

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were analyzed. These samples belonged to mothers who declared heavy MA abuse during pregnancy. Hence, high concentrations of AP and MA were expected in meconium samples and only 0.3-

Figure 4. SIM chromatogram of an extract of sample 20014 containing 0.038 µg/g AP and 0.226 µg/g MA.

0.5-g samples were submitted to the extraction and analysis procedure. All the analyzed samples contained high concentration, of both MA and AP, without the presence of any methylendioxy

derivative (Figure 4 and Table 4). Furthermore, it has to be remarked that these last samples were over 1 year old and have been stored at -20 °C. The good comparison between the results Analytical Chemistry, Vol. 76, No. 7, April 1, 2004

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Table 4. Analyte Concentration in Meconium Samples Positive for AP, MA, and MDMA sample

AP (µg/g)

MA (µg/g)

MDMA (µg/g)

569 19235b 19338b 19574b 19620b 19748b 19805b 20014b

nda 0.676 (0.798) 0.449 (0.470) 0.537 (0.582) 0.908 (1.000) 0.139 (0.153) 1.019 (1000) 0.038 (0.040)

nd 1.024 (>1000) 1.102 (>1000) 1.057 (>1000) 0.945 (>1000) 0.960 (>1000) 1.157 (>1000) 0.226 (0.204)

0.012 nd nd nd nd nd nd nd

a nd, not detected. b Samples donated by Dr. Christine Moore; in parentheses, values obtained at U.S. Drug Testing.

we obtained with those obtained when they were first analyzed by Dr. Moore by GC/MS electron impact shows the excellent stability of AP and MA in meconium. CONCLUSIONS The LC-MS method to analyze amphetamine derivatives in meconium reported in this paper was validated according to (24) Global Illicit Drug Trends 2000; United Nations Office for Drug Control and Crime Prevention, United Nation Publication: New York, 2000; pp 1016. (25) Ho, E.; Karimi-Tabesh, L.; Koren G. Neurotoxicol. Teratol. 2001, 23, 561567.

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internationally accepted criteria.22,23 The method simultaneously detects amphetamine, methamphetamine, 3,4-methyledioxyamphetamine, 3,4-methylenedioxymethamphetamine, 4-hydroxy-3methoxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine in human fetus. Indeed, the method is sensitive enough for determination of amphetamine derivatives in the range of nanograms using 1 g of meconium sample. The accurate assessment of fetal exposure to drugs of abuse through the objective measure of biomarkers in meconium, which is a repository of substances to which the fetus is repeatedly exposed in utero, could be of major importance since it provides the basis for appropriate treatment and adequate follow-up of exposed newborns. ACKNOWLEDGMENT This study was supported by “Area Progetto Droga” (Convenzione 513A/4) from Istituto Superiore di Sanita`, Roma, Italy. The authors thank Dr. Christine Moore for her generous cooperation and all suggestions given during methodology development.

Received for review December 2, 2003. Accepted January 22, 2004. AC035419X