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Journal of Analytical Toxicology, Vol. 34, July/August 2010

Famprofazone Use Can Be Misinterpreted as Methamphetamine Abuse Kuei-Hui Chan1,*, Mei-Chich Hsu2, Chen-Yu Tseng3, and Wei-Lan Chu3 1Graduate

Institute of Coaching Sciences and 2Graduate Institute of Sports Sciences, National Taiwan Sport University, 250 Wen Hua 1st Road, Kweishan, Taoyuan 33301, Taiwan and 3Taipei Veterans General Hospital, Division of Clinical Toxicology, Department of Internal Medicine, Taipei, Taiwan

Abstract Famprofazone, a major ingredient of Gewolen®, is an analgesic that has been demonstrated to be metabolized to methamphetamine (MA) and amphetamine (AM) following administration. Therefore, a famprofazone user may be interpreted as an illicit MA abuser in Taiwan because the user’s urine tested positive for MA. The purpose of this study was to investigate whether the concentration of MA metabolized from a single dose of Gewolen users would offend the official controlled substance regulation and be identified as MA-positive. Subjects (n = 6) received 25 mg of famprofazone and collected all urine specimens at certain timed intervals for 48 h after drug administration. The urine specimens were screened by immunoassay and then confirmed by gas chromatography–mass spectrometry (GC–MS). The highest concentration of amphetamines by immunoassay was 1954 ng/mL, and 18.8% of the urine specimens’ amphetamines concentrations exceeded 500 ng/mL. The MA and AM concentrations by GC–MS analysis of these urine specimens ranged from 901 to 2670 ng/mL and 208 to 711 ng/mL, respectively. These urine specimens were interpreted as MApositive (≥≥ 500 ng/mL MA and ≥ 100 ng/mL AM), according to the official test methods of Taiwan. The MA positive results appeared within 2–34 h. It is therefore clearly possible to misinterpret the legitimate famprofazone user as an MA abuser in Taiwan.

Introduction Famprofazone (3-[N-methyl-(α-methyl-β-phenyl)-ethylaminomethyl]-4-isopropylnorantipyrine) is an analgesic that has been demonstrated to be metabolized to methamphetamine (MA) and amphetamine (AM) following administration (1,2). After administration of racemic famprofazone, the R-(2)-forms of MA and AM are excreted in urine (2). MA (R,SN-methyl-1-phenyl-2-propanamine) and AM (R,S-1-phenyl-2propanamine) are powerful stimulants of the central nervous system. They are used as legitimate medicaments, drugs of abuse, or doping agents in sports (2). After MA use, both MA * Author to whom correspondence should be addressed. E-mail: [email protected].

and AM are excreted in the urine (3). Therefore, the detection of urinary MA and AM in forensic analysis can be an indicator of illicit MA abuse. However, an MA-positive result may be caused by famprofazone administration. Two legally and locally manufactured brand-name medicines (Gewolen tablet and Paisao® capsule) containing famprofazone are found on the market. Each Gewolen tablet (433 mg) contains 25 mg of famprofazone, 200 mg of acetaminophen, 75 mg of isopropyphenazone, and 25 mg of caffeine. Each Paisao capsule contains 25 mg of famprofazone, 250 mg of acetaminophen, 75 mg of isopropyphenazone, and 30 mg of caffeine anhydrous. Both drugs are used for pain relief, and the recommended Gewolen usage for an adult is one tablet (25 mg of famprofazone) per dose and twice per day (4). Studies showed that after the administration of 50 mg of famprofazone, the urinary concentrations of MA and AM exceeded 500 ng/mL and 100 ng/mL, respectively (5–8). MA and AM, both strong central nervous system stimulants, are classified as a Schedule 2 drug under the Controlled Drugs Act and are listed as restricted ingredients that cannot be manufactured or traded in Taiwan (9). MA and AM are considered illicit drugs in most of the drug-abuse urine-testing laboratories in Taiwan. Current practice requires the use of two assays based on different analytical principles. First, immunoassays are frequently used for urine screening in order to differentiate between negative and presumptively positive samples. Presumptively positive results (amphetamines screen ≥ 500 ng/mL) must be confirmed by a second independent method, such as gas chromatography–mass spectrometry (GC– MS). For the official “truly positive” MA test, the Food and Drug Administration (FDA) in Taiwan instructed its certified drug-abuse urine-testing laboratories that at least 100 ng/mL of AM must be present in urine specimens that are positive for MA ≥ 500 ng/mL (10). Therefore, distinguishing the differences in MA and AM in urine between famprofazone users and MA abusers is an important issue (11). The d-isomer of MA is a serious illicit drug because it is more potent in psychostimulating activities than l-isomers (12). Chan et al. (13) demonstrated that the urinary d-enantiomers of MA and AM from MA abusers in Taiwan were 97.5 ±

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1.7% and 98.6 ± 2.1%, respectively. Furthermore, the MA and AM metabolized from famprofazone are both the d- and the lenantiomers. Studies showed that 70–100% of l-MA and 50– 73% of l-AM were presented in urine after famprofazone administration (6,7,14). Detection of the enantiomeric composition of MA and AM is very valuable in determining the involvement of this drug. Famprofazone is also converted to several other metabolites, including 3-hydroxymethylpyrazolone, which is subsequently excreted in the urine (15). One study (1) suggested that detection of 3-hydroxymethylpyrazolone or other components together with the concentrations and enantiomeric composition of the MA and AM in the sample allows the determination of the use of famprofazone. From a forensic standpoint, it is important to consider that the precursor drugs are controlled and dispensed by prescrip-

tion. Unfortunately, Gewolen and Paisao can be purchased in drugstores without a prescription, and thus Gewolen and Paisao users may lack valid medical prescriptions to help explain the MA-positive urinary result. Moreover, there are no Medical Review Officers to review laboratory results and determine the possibility of valid medical use of the drug in Taiwan. Although the enantiomeric composition of MA and AM is a useful method, the detection of enantiomers is not a routine test in Taiwan. Therefore, a famprofazone user may be interpreted as an illicit MA abuser because he or she tested positive for both MA and AM. Several studies have investigated the urinary concentrations of MA after administering 50 mg of famprofazone (5–8,14). However, the recommended dose of Gewolen is only one tablet (25 mg of famprofazone). Moreover, most of the studies (5– 7,14) did not conduct the immunoassay screen prior to GC–MS confirmation. Yoo et al. (8) presented a study with an initial enzyme multiple immunoassay test (EMIT) d.a.u. amphetamines assay followed by GC–MS to analyze the urinary MA concentration following 50 mg of famprofazone administration. It showed that all urine specimens within 46 h after famprofazone administration presented the positive result of EMIT (change in optical density > 43). However, not all of these urine specimens had a positive result with the GC–MS test. The purpose of this study was to investigate the possibility of being urine MA-positive after the administration of 25 mg of famprofazone. Figure 1. Typical gas chromatogram of amphetamine-d5-HFBA (peak 1); amphetamine-HFBA (peak 2); The official test methods of Taiwan were methamphetamine-d11-HFBA (peak 3); and methamphetamine-HFBA (peak 4). The concentration of all used to interpret the drug test results. substances is 250 ng/mL.

Materials and Methods Chemicals and reagents

Figure 2. Typical gas chromatogram of l-amphetamine-d5-l-TPC (peak 1); l-amphetamine-l-TPC (peak 2); d-amphetamine-d5-l-TPC (peak 3); d-amphetamine-l-TPC (peak 4); l-methamphetamine-d11-l-TPC (peak 5); l-methamphetamine-l-TPC (peak 6); d-methamphetamine-d11-l-TPC (peak 7); and d-methamphetamine (peak 8). Concentration were 250 ng/mL for peaks 2, 4, 6, and 8 and 5000 ng/mL for peaks 1, 3, 5, and 7.

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Standard solutions of 1 mg/mL of dlMA, R(–)-MA, S(+)-MA, dl-AM, R(–)-AM, and S(+)-AM in methanol and methanolic deuterated standards of dl-MA-d11 and dlAM-d5 were purchased from Cerilliant (Austin, TX). The derivatizing reagents, heptafluorobutyric anhydride (HFBA) and N-trifluoroacetyl-l-prolyl chloride (l-TPC), were purchased from Macherey-Nagel (Duren, Germany) and Sigma Aldrich (Deisenhofen, Germany), respectively. Gewolen tablets (each containing 25 mg of famprofazone, 200 mg of N-acetyl-paminophenol, 75 mg of isopropylantipyrine, and 25 mg of caffeine) were purchased over-the-counter in Taiwan. Double-deionized water was obtained from a Millipore Direct-Q5 system (Bedford,

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MA). Other chemicals were purchased from Merck (Darmstadt, Germany). Drug administration and urine collection

Six untrained healthy volunteers (1 female and 5 males) with a mean age (± SD) of 25.2 (± 1.9) years, height of 174.5 (± 7.7) cm, and body mass of 79.0 (± 14.1) kg participated in the experiment. Subjects administered one tablet of Gewolen with 200 mL of water 30 min after breakfast. Urine specimens were collected in polyethylene bottles in the intervals of 0–1, 1–2, 2–3, 3–4, 4–5, 5–6, 6– 7, 7–8, 8–9, 9–12, 12–15, 15–22, 22–28, 28–34, 34–40, and 40–48 h after administering the drug. To ensure sufficient urine flow during the sampling period, 100 mL of water was ingested at 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, and 15 h on treatment day. The pH, volume, and specific gravity of each urine specimen at room temperature were recorded, and the specimen was stored at –20°C before sample preparations for screening and chromatographic analysis. Approval for the study’s procedures was granted by the Committee for Research on Human Subjects of Da Yeh University. Analytical procedure for MA and AM

Two hundred microliters of urine specimens were used to screen the amphetamines by a HITACHI - 7600 automatic screening analyzer (Tokyo, Japan) and DRI ® amphetamines immunoassay reagents (Microgenic, Fremont, CA) kits. After the screening, the urine specimens were then analyzed by GC–MS with solid-phase extraction (SPE) to confirm the concentrations of MA and AM. Fifty microliters of MA-d11 (5 µg/mL), 50 µL of AM-d5 (5 µg/mL), and 1 mL of 100 mM phosphate buffer solution (pH 6.0) were added to 0.5 mL of the urine specimens. The sample solution was passed through a HCX SPE cartridge (130 mg, 3 mL) as described by Internal Sorbent Technology (Hengoed, U.K.). MA and AM were eluted with 3 mL of CH2Cl2/IPA/NH4OH (78:20:2, v/v/v). The extraction solution was evaporated to dryness under a gentle stream of nitrogen gas at room temperature. The residue was added to 100 µL of ethyl acetate and 25 µL of HFBA and incubated for 20 min at 70°C.

Table I. Urinary pH and Amphetamines, Methamphetamine (MA), and Amphetamine (AM) Concentrations Following Administration of 25 mg of Famprofazone

Hours Post Dose

pH

Immunoassay Screening Amphetamines (ng/mL)

GC–MS Confirmation MA AM concentration concentration (ng/mL) (ng/mL)

% l-MA

% l-AM

Subject A Pre-dose 0–1 1–2 2–3 3–4 4–5† 5–6 6–7† 7–8 8–9 9–12 12–15 15–22 22–28 28–34† 34–40 40–48

5.54 5.27 5.95 6.98 7.18 6.25 6.27 5.71 5.75 6.50 5.93 6.81 6.33 6.81 5.29 6.24 6.04

116 43 29 90 294 842 281 621 454 143 341 206 275 210 614 195 129

–* – 91 166 484 2753 541 1167 881 324 670 381 523 373 1141 275 188

– – – 37 115 407 131 268 204 76 159 96 130 94 278 75 50

– – 68.24 68.04 68.59 69.93 68.57 69.64 70.17 68.56 70.75 69.96 70.24 70.75 73.05 72.52 73.55

– – – – 55.99 57.19 55.98 57.16 57.35 69.43 57.78 60.01 57.64 58.32 61.87 61.23 –

Subject B Pre-dose 0–1 1–2 2–3† 3–4† 4–5† 5–6† 6–7 7–8 8–9† 9–12 12–15† 15–22† 22–28 28–34 34–40 40–48

6.89 6.80 6.88 6.29 6.35 5.88 5.90 6.50 6.56 6.12 5.46 5.23 5.28 5.51 6.30 6.76 6.10

73 48 254 1076 726 785 587 164 257 510 410 640 843 343 100 93 126

– 51 461 1754 1357 1455 1051 336 486 954 800 1056 1418 593 134 98 125

– – 120 424 344 376 288 104 153 289 249 372 480 228 61 – –

– 63.23 68.14 70.27 71.24 71.68 71.78 73.68 71.02 72.77 73.70 75.88 75.09 78.44 77.59 81.14 83.19

– – 56.02 55.23 54.61 54.46 54.23 54.72 53.36 54.84 53.98 54.30 54.33 55.55 55.73 – –

Subject C Pre-dose 0–1 1–2 2–3 3–4 4–5 5–6† 6–7 7–8 8–9 9–12

5.66 5.67 6.06 6.79 6.33 5.96 5.60 6.56 6.64 6.41 6.36

88 37 276 196 299 345 1954 178 61 71 92

– 60 590 365 687 671 2760 497 158 273 370

– – 143 98 175 172 711 137 40 74 110

– 68.54 68.27 68.25 68.63 69.19 69.87 69.32 69.31 69.38 70.15

– – 58.21 61.65 56.26 69.48 59.01 55.55 – 55.82 55.66

Table I continued on next page * Indicates concentration below LOQ. † An MA-positive result according to the official analytical methods in Taiwan.

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Table I. Urinary pH and Amphetamines, Methamphetamine (MA), and Amphetamine (AM) Concentrations Following Administration of 25 mg of Famprofazone (Continued) Immunoassay Screening Amphetamines (ng/mL)

GC–MS Confirmation MA AM concentration concentration (ng/mL) (ng/mL)

Hours Post Dose

pH

12–15 15–22 22–28 28–34 34–40 40–48

6.93 6.17 5.81 6.87 6.69 6.82

117 208 147 53 76 45

183 393 415 107 113 85

Subject D Pre-dose 0–1 1–2† 2–3 3–4 4–5 5–6 6–7 7–8 8–9 9–12 12–15† 15–22 22–28† 28–34 34–40 40–48

5.79 5.37 6.37 7.38 6.93 6.85 6.37 7.25 7.32 7.17 6.77 5.68 6.44 5.67 6.94 6.66 6.28

108 171 865 127 113 344 182 115 66 66 148 759 257 593 119 100 111

Subject E Pre-dose 0–1 1–2† 2–3 3–4 4–5 5–6 6–7 7–8 8–9 9–12 12–15 15–22 22–28 28–34 34–40 40–48

6.25 5.97 7.02 7.05 6.98 6.38 7.02 7.42 7.46 6.98 6.57 5.55 6.13 6.08 7.18 5.57 6.12

Subject F Pre-dose 0–1 1–2 2–3

5.39 5.31 5.95 6.95

% l-MA

% l-AM

51 127 136 34 38 –

70.56 71.28 72.26 73.43 74.14 75.01

55.39 58.84 56.78 –* – –

– 18 1627 244 220 608 356 174 119 153 295 1316 342 929 153 70 103

– – 325 61 59 153 100 54 37 51 93 408 130 363 76 42 68

– 61.00 69.88 69.59 69.84 71.01 70.95 70.95 70.77 71.73 73.00 74.64 76.34 77.95 78.42 78.56 81.59

– – 58.20 53.93 54.56 54.70 54.47 54.24 – 54.28 55.25 54.08 54.40 55.73 55.56 – 57.16

144 184 676 423 237 269 145 65 92 88 238 350 354 214 95 251 111

– 133 1104 708 459 466 214 98 102 168 467 597 580 370 86 337 134

– 33 208 146 99 105 54 23 23 42 110 144 145 102 24 96 43

– 61.59 68.53 68.69 68.65 68.92 67.95 67.67 67.37 68.17 69.71 71.14 71.17 72.53 72.29 76.01 76.45

– – 54.82 53.49 53.26 55.65 53.27 – – 53.48 54.02 53.95 55.30 56.76 – 57.37 57.49

108 157 265 254

– – 147 382

– – 30 79

– – 68.10 68.71

– – – 55.81

Table I continued on next page * Indicates concentration below LOQ. † An MA-positive result according to the official analytical methods in Taiwan.

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The solution was cooled to room temperature and evaporated to dryness under a gentle stream of nitrogen gas at room temperature and reconstituted in 50 µL of ethyl acetate prior to GC–MS (Agilent 6890N/5973N, Santa Clara, CA) analysis. The GC conditions were as follows: J&W DB-5 column (30 m × 0.25mm i.d., 0.25-µm film thickness, Agilent); column temperature 70°C (1 min) programmed to 180°C at 20°C/min for 1 min and then programmed to 230°C at 35°C/min for 1 min; injection port and interface temperatures at 200 and 280°C, respectively; injection splitless mode; and helium carrier gas (1.1 mL/min). The MS conditions were ionization mode, electron impact; ionization energy, 70 eV; and monitored ions, m/z 254, 118, 210 for MA, m/z 260, 213 for MA-d11, m/z 240, 91, 118 for AM, and m/z 244, 123 for AM-d5. (Target ions used for quantification are underlined.) A blank urine specimen was collected from a non-drug user. None of the drug in urine was detected by full-scan GC– MS. The blank human urine was spiked with MA and AM at the concentrations of 15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 ng/mL for quantitative comparisons. Other quality control samples (250, 500, and 750 ng/mL) were prepared in the same way for precision evaluation. Pharmacokinetic parameters for MA and AM were calculated. The time to reach maximum concentration (Tmax) and maximum urinary excretion rate [(dXu/dt)max] were directly determined from the resulting concentration-time profiles. The elimination rate constant was calculated from log-linear leastsquares regression of the terminal phase data points. The terminal elimination half-life (t½) was calculated as 0.693 divided by the elimination rate constant. Non-compartmental model was applied to calculate the area under the urine concentration-time curve from 0 to 48 h (AUC0–48), which was determined by the linear trapezoidal rule. The program used for the determination of these pharmacokinetic parameters was Kinetica software version 5.0 (ThermoFisher Scientific, Waltham, MA). Analytical procedure for d- and l-MA and d- and l-AM

A GC–MS method described by Green-

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hill et al. (6) was applied to separated the d- and l-enantiomers of MA and AM at a final concentration of 250 ng/mL. The of MA and AM. One-hundred microliters of MA-d11 (5 µg/mL) retention times of l-AM-d5, l-AM, d-AM-d5, d-AM, l-MA-d11, and 100 µg of AM-d5 (5 µg/mL) were added to 2 mL of the l-MA, d-MA-d11, and d-MA were 15.62, 15.68, 15.98, 16.04, urine specimens. Extraction was accomplished by adding 2 mL 18.04, 18.16, 18.36, and 18.48 min, respectively. The linof 1 M NaOH and 4 mL ethyl acetate. After shaking and cenearity of the assay over the range of 7.8125–500 ng/mL and trifugation, the top organic layer was transferred to a clean, dry 15.625–500 ng/mL for MA and AM showed the correlation coglass tube followed by adding 50 µL of l-TPC and incubating at efficient of r2 > 0.9999. The limits of detection for d-MA, room temperature for 15 min. Three milliliters of 0.01 M NaOH were then added and shaken for 10 min. After Table I. Urinary pH and Amphetamines, Methamphetamine (MA), and centrifugation, the top organic layer was Amphetamine (AM) Concentrations Following Administration of 25 mg of Famprofazone (Continued) transferred and evaporated under a stream of nitrogen at 50–60°C. The GC–MS Confirmation residue was reconstituted in 50 µL of Immunoassay ethyl acetate and injected into the GC– Screening MA AM MS. Instrumental conditions were as folHours Amphetamines concentration concentration lows: splitless injection; injector and inPost Dose pH (ng/mL) (ng/mL) (ng/mL) % l-MA % l-AM terface temperature at 270°C; oven 3–4 6.56 207 337 73 68.56 55.39 temperature 130°C for 6 min, increasing 4–5 6.26 230 400 88 68.91 57.15 to 220°C at 6°C/min, holding for 5 min. 5–6 5.88 320 540 118 70.02 54.74 Ions monitored were m/z 237, 241, 251, 6–7† 6.10 651 1102 233 69.73 55.06 260 for d- and l-AM; d,l-AM-d5; d- and l7–8† 6.06 1108 1806 379 70.71 54.83 MA; d,l-MA-d11, respectively. Each ana8–9 6.36 163 304 76 70.06 51.92 lytical batch of samples was calibrated at 9–12 5.96 340 591 139 70.94 54.80 250 ng/mL and analyzed with control 12–15† 6.17 565 901 214 71.67 59.86 15–22 6.49 410 698 174 72.04 54.49 samples at 7.8125, 15.625, 31.25, 62.5, 22–28 5.90 406 676 182 74.15 54.69 125, 250, and 500 ng/mL. 28–34 34–40 40–48

Results and Discussion

6.99 6.35 5.63

89 211 281

115 245 377

37 78 126

73.83 76.52 78.03

–* 55.42 56.21

* Indicates concentration below LOQ. † An MA-positive result according to the official analytical methods in Taiwan.

Reliability of the method

Figure 1 illustrates the chromatogram obtained from drug-free urine spiked with both MA and AM at a final concentration of 250 ng/mL. The retention times of AM-d 5, AM, MA-d 11, and MA were 6.38, 6.40, 7.05, and 7.08 min, respectively. The linearity of the assay over the range of 15.625–2000 ng/mL showed correlation coefficients of r2 = 0.9998 and 0.9999 for MA and AM, respectively. The limits of quantification and detection were 15.625 ng/mL. The intraday precision and accuracy obtained at three concentration levels (250, 500, and 750 ng/mL) were 0.73–1.62% and –11.30– 19.78%, respectively. The interday precision and accuracy were 0.94–1.33% and –12.16–17.67%, respectively. These results demonstrated that this GC–MS method was suitable for the quantification of MA and AM in urine with satisfactory accuracy and precision. Figure 2 illustrates the chromatogram obtained from drug-free urine spiked with d- and l-enantiomers

Table II. Urinary Pharmacokinetic Parameters for Methamphetamine and Amphetamine Following Administration of 25 mg of Famprofazone Subject

Tmax (h)*

Methamphetamine A 6.5 B 1.5 C 1.5 D 5.5 E 13.5 F 5.5 Mean (± SD) 5.7 (± 4.4) Amphetamine A B C D E F Mean (± SD)

6.5 1.5 1.5 5.5 13.5 5.5 5.7 (± 4.4)

(dXu/dt)Max (µg/h)*

t1/2 (h)*

AUC0-48 (µg/mL·h)*

107.4 112.0 176.4 121.4 124.0 149.0 131.7 (± 26.2)

14.7 11.5 8.6 7.0 10.0 13.0 10.8 (± 2.8)

1402.2 1473.3 1604.1 1449.6 1549.5 1613.3 1515.3 (± 86.6)

24.7 29.2 42.8 34.1 29.9 32.6 32.2 (± 6.1)

15.9 11.7 5.2 9.8 11.4 16.7 11.8 (± 4.2)

330.2 472.0 441.5 509.6 384.4 406.0 423.9 (± 64.2)

* Tmax = time to reach maximum concentration; (dXu/dt)max = maximum urinary excretion rate; t1/2 = terminal elimination half-life; AUC0-48 = area under the urine excretion rate-time curve for the 48 h after dose.

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l-MA, d-AM, and l-AM were 7.8125, 7.8125, 15.625, and 15.625 ng/mL, respectively. Urine analysis of the famprofazone users

Table I illustrates the urinary pH, immunoassay, and GC–MS concentration as well as l-enantiomer composition of MA and AM after the administration of 25 mg famprofazone. The pH value of the urine specimens ranged from 5.23 to 7.46. The composition of l-MA and l-AM were 71.43 ± 3.81% (mean ± SD) and 56.21 ± 2.99%, respectively. This result is consistent with previous single-dose (6,7) and multiple-dose studies (14). The highest concentration of amphetamines was 1954 ng/mL, which appeared 5–6 h after administration. However, the amphetamines’ concentrations in most of the urine specimens were lower than 1000 ng/mL. Screening by immunoassay within 48 h after famprofazone administration showed that 18.8% (18 out of 96) of the urine specimens’ amphetamines concentrations exceeded the cutoff value (500 ng/mL), which ranged from 510 to 1954 ng/mL. The MA and AM concentrations [mean ± SD (range)] analyzed by GC–MS of these urine specimens were 1425 ± 555 (901–2760) ng/mL and 354 ± 117 (208–711) ng/mL, respectively. Data showed that all of these urine specimens were confirmed ≥ 500 ng/mL MA and ≥ 100 ng/mL AM. Thus, the 18.8% urine specimens will be considered as illicit MA abuse. The result of our study did not agree with Yoo et al. (8), whose data showed that the positive result of EMIT would not be confirmed by GC–MS. This may be due to the differences of dosage administration and/or immunoassay kits. After one tablet of Gewolen administration, MA-positive results appearing at 2 to 34 h varied with different individuals (Table I). Nevertheless, the average concentrations of MA and AM from 6 subjects demonstrated that MA-positive

results appearing between 2 and 8 and 12 and 28 h after administration (Figure 3). Studies of Oyler et al. (16) showed the peak MA and AM concentrations [mean ± SD (range)] after 10 mg MA administration were 2310–14,660 (4720 ± 2302) ng/mL and 264–660 ng/mL, respectively. This study showed that the MA concentrations in urine after the administration of 25 mg famprofazone were far lower than those concentrations in the urine of 10 mg MA users, but the AM concentration was similar. Evaluating the concentration of MA in the urine can also be very informative. Mean Tmax for both MA and AM was 5.7 ± 4.4 h (mean ± SD) (Table II). Estimated (dXu/dt)max were 131.7 ± 26.2 µg/h in MA and 32.2 ± 6.1 µg/h in AM, respectively. Mean t½ for MA and AM were 10.8 ± 2.8 h and 11.8 ± 4.2 h, respectively. Mean AUC 0–48 was 1515.3 ± 86.6 µg/mL·h in MA and 423.9 ± 64.2 µg/mL·h in AM, respectively. Data showed that the individuals had a great variation in the time of amphetamines metabolite excretion after 25 mg of famprofazone administration. Kim et al. (17) investigated the urinary pharmacokinetics of MA and AM following 10 mg and/or 20 mg MA oral administration. The mean MA t½ for 10 and 20 mg MA oral administration was 22.6 ± 7.2 h (range 15.3–34.9 h) and 25.1 ± 6.0 h (range 20.1– 32.7 h), respectively. Meanwhile, the mean AM t1⁄2 for 10 and 20 mg MA oral administration was 19.8 ± 7.3 h (range 12.3–30.7 h) and 22.0 ± 8.0 h (range 13.6–31.5 h), respectively. The work by Oyler et al. (16) also showed the peak MA and AM concentrations after 10 mg MA administration occurring within 1.5– 60 h. Therefore, the elimination half-life and Tmax of MA and AM after 25 mg famprofazone administration were shorter than in those of MA users. The results indicated that the administration of one tablet of Gewolen definitely produced an MA-positive results under the official test methods of Taiwan. Oh et al. (19) indicated that at least 20% dose of famprofazone would break down to MA in the plasma, which could have significant clinical implications. The medication of famprofazone has been banned in Korea (8), and it is also listed as a banned substance under the class of stimulant by the World Anti-Doping Agency (WADA) to prohibit athletes using it in competition since 2005 (18). The Department of Health in Taiwan should consider withdrawing the medication of famprofazone.

Conclusions

Figure 3. Time courses of urinary concentrations of methamphetamine and amphetamine after administration of 25 mg famprofazone. Data are expressed as mean ± SD (n = 6).

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This study investigated the metabolite results of amphetamines, MA and AM, following the administration of 25 mg of famprofazone as well as interpreted the results according to the forensic rule of Taiwan. The results showed that 18.8% of the urine specimens within 48 h after 25

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mg of famprofazone administration presented the amphetamines-positive result through immunoassay (≥ 500 ng/mL). The MA and AM concentrations of these urine specimens were confirmed ≥ 500 ng/mL MA and ≥ 100 ng/mL AM by GC–MS analysis. Thus, the administration of one tablet of Gewolen would result in misinterpretation of illicit MA abuse, according to the official analytical methods in Taiwan.

Acknowledgments Financial support provided by the National Science Council (NSC) of the Executive Yuan, Taiwan under grant No. NSC 94-2413-H-252-003 is gratefully acknowledged.

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Manuscript received December 7, 2009; revision received February 11, 2010.

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