Urinary Excretion Profiles of Two Major Triazolam Metabolites ...

Journal of Analytical Toxicology, Vol. 29, May/June 2005

Urinary Excretion Profilesof Two Major Triazolam Metabolites ( -Hydroxytriazolam and 4-Hydroxytnazolam 9

!

Kenji Tsujikawa*, Kenji Kuwayama, Hajime Miyaguchi, Tatsuyuki Kanamori, Yuko Iwata, Hiroyuki Inoue and Tohru Kishi National ResearchInstituteof Police Science6-3-1, Kashiwanoha,Kashiwa, Chiba 277-0882,Japan

Abstract The objective of this study was to examine urinary excretion profiles of two major triazolam metabolites, ~hydroxytriazolam (~OHTRZ) and 4-hydroxytriazolam (4-OHTRZ) in humans. Urine samples were collected from three healthy male volunteers who had been previously administered single 0.25- and 0.5-rag dosesof lriazolam 24 h and 48 h, respectively,before sample collection. After enzymatic hydrolysisand extraction, each sample was analyzed by liquid chromatography-massspectrometry, u.-OHTRZ was rapidly excreted, with the maximum concenlrations appearing in the first or second sample collected after ingestion, with the majority of the drug being excreted within 12 h. Meanwhile, 4-OHTRZ was excreted more slowly than ~-OHTRZ. The ~OHTRZ/4-OHTRZ ratios were initially greater than 19.7, then decreased rapidly, reaching a nearly constant value for times in excessof 12 h.

victims were examined with a mean delay of 17.5 h after rape cases in a Paris suburb. Therefore, it is important to determine the excretion profile of hypnotic drugs or their metabolites in urine. Urinary excretion profiles of other benzodiazepine hypnotics, flunitrazepam and diazepam, were determined by gas chromatography-mass spectrometry (4,5). However, triazolam has not been examined using such chromatographic methods. In this study, we report on the urinary excretion profiles of mOHTRZ and 4-OHTRZ in a clinical dosage range. The analytical method is based on the enzymatic hydrolysis of the glucuronide-conjugated metabolites, followed by quantification by liquid chromatography-mass spectrometry (LC-MS).

Experimental Materials

Introduction

Triazolam is a triazolobenzodiazepine derivative and one of the most frequently prescribed hypnotics in Japan. It is a potent hypnotic with a short duration of action in humans and is administrated orally at doses of 0.125-0.5 mg. Triazolam is metabolized to two major metaboIites, cr (cr OHTRZ) and 4-hydroxytriazolam (4-OHTRZ) by the human CYP3A4 (1). After a single oral dose of 14C-triazolam, the excretion of cr and 4-OHTRZ accounted for 69% and 11% of the urinary 14C-activity,most of which was excreted in a glucuronide conjugated form, whereas only 2.1% was excreted in the form of unchanged triazolam (2). Because hypnotic drugs, including triazolam, have been alleged to be involved in date-rape cases, analysis of their urinary metabolites is an important forensic issue. However, an examination may not always be performed soon after the case is reported. In fact, Marc and colleagues (3) reported that * Author to whom correspondence should be addressed. E-mail [email protected]

240

c~-OHTRZ was obtained from Sigma (St. Louis, MO). 4-OHTRZ and nitrazepam were supplied by Upjohn Co. (Kalamazoo, MI). 13-Glucuronidase from E. cell, Type IX-A (No. G 7396), was obtained from Sigma. The activity of the ~-glucuronidase used in this study was determined by the method reported by Fishman et al. (6). All other chemicals used in the experiments were of analytical grade. Subjects, doses, and urine collection The study protocol was approved by the Institutional Review Board. Three healthy male volunteers (Mongoloid, ages:27--45) participated after giving their informed consent. Triazolam (Halcion, Pharmacia K.K.) was administered orally in the form of a single dose (0.25 mg or 0.5 rag) to each subject at 11 p.m.-12 a.m. Urine samples were collected at random time points up to 24 h (for a 0.25-rag dose) or 48 h (for a 0.5-rag dose) after ingestion. The total number of urine samples was 5-13 in each experiment. The volume of all urine samples was measured, and aliquots were stored at 20~ until used for the analysis. The washout period between experiments was at least one week for each individual.

Reproduction(photocopying)of editorial contentof this journal is prohibitedwithout publisher'spermission.

Journal of Analytical Toxicology,Vol. 29, May/June2005

Hydrolysis of glucuronide-conjugates and extraction temperature, 300~ MS data were collected in the selected Hydrolysis of glucuronide-conjugated triazolam metaboion monitoring mode with a dwell time of 200 ms per monilites and the extraction procedure followed previously retoring ion. Analytical detection was based on the monitoring of ported methods (7). A 1-mL urine sample was adjusted to pH the [M+H]§ protonated molecular ions of ~-OHTRZ and 6.8 by adding 1 mL of 0.2M phosphate buffer (pH 6.8), fol4-OHTRZ (m/z 359), as well as nitrazepam (m/z 282). lowed by 100 U of ~-glucuronidase. After incubation at 37~ for 90 rain, the mixture was made alkaline by the addition of 3M sodium hydroxide solution, and I mL of 0.2M carbonate Results buffer (pH 10.5) was then added. (~-OHTRZ and 4-OHTRZ were extracted twice with 3 mL of a mixture of hexane/ dichloromethane (50:50, v/v) for 5 rain. After centrifugation at Analytical methodology A typical LC-MS chromatogram obtained from an extract of 3000 rpm for 5 rain, the organic layer was transferred to a a urine sample is shown in Figure 1. In the preliminary study, tapered glass tube and evaporated to dryness under a stream of nitrogen. The residues were reconstituted in 100 IJLof LC-MS mobile phase containing Table I. Recoveries of a-OHTRZ and 4-OHTRZ From Urine* 1 pg/mL nitrazepam as an internal standard ~-OHTRZ 4-OHTRZ and a 20-pL aliquot was used in the analysis. Tests to determine the recovery, precision, Concentration (ng/mL) Recovery(%) Concentration(ng/mL) Recovery(%) and accuracy of the method were performed 95.0• 20 91.2• by using blank urine spiked with (z-OHTRZ 3oo 97.6• 8 92.3• and 4-OHTRZ. The concentrations spiked 4o 97.4• 2 92.5• were as follows: 300, 40, and 4 ng/mL for 4 mOHTRZ and 20, 8, and 2 ng/mL for 4* n = 5, mean• standarddeviation. OHTRZ. The recoveries were calculated by comparing the ratios of the peak areas for each analyte after sample extraction to stanTable II. Summary of Analyte Accuracy and Precision dards prepared in a urine matrix (standards spiked into blank urine after extraction). The Nominal Concentration (ng/mL) accuracy of the assaywas evaluated by percent u.-OHTRZ 4-OHTRZ deviation (%DEV) from the nominal concen300 40 4 20 8 2 tration using the formula: [%DEV = 100 x (mean back calculated concentration - nomAccuracy (n = 5) inal concentration)/nominal concentration]. Mean observed concentration (ng/mL) 275.4 45.2 4.24 21.8 8.65 2.00 Intra- and interassay precision is expressed %DEV -8.2 13.0 6.0 9.0 8.1 0.0 as the coefficient of variation (CV) of the experimental values at each concentration. Precision Apparatus and chromatographic conditions The separation and detection of free triazolam metabolites were carried out using a Waters LC-MS system (Milford, MA) consisting of a 2690 series high-performance LC (solvent degasser, pump, autosampler and column oven) and a ZQ single-quadrupole MS equipped with an electrospray ionization (ESI) interface. Chromatographic separations were performed with a Mightysil RP-18 column (150 x 2.0-ram i.d., 5 1Jm,Kanto Chemical, Tokyo, Japan), maintained at 40~ The mobile phase was 50ram ammonium acetate (pH 4.0)/ methanol (57:43, v/v) pumped at a flow rate of 0.2 mL/min. The positive ion mode of ESI-MS was used for all measurements. The optimized parameters were as follows: cone voltage, 55 V; capillary voltage, 4000 V; cone gas flow, 50 L/h; desolvation gas flow,350 L/h; and desolvation

Intra-assay(CV %, n = 5) Interassay(CV %, n = 5)

3.4 6.1

3.8 7.1

2.4 6.8

3.0 6.4

1.9 7.8

3.9 10.4

Table III. Urinary Recovery of ~OHTRZ and 4-OHTRZ after the Oral Administration of Triazolam (0.25 mg or 0.5 rag) to Healthy Volunteers (n = 3) Colledion Interval (h)

Subjed A

Subject B

~OHTRZ* 4-OHTRZ* ~OHTRZ* 4-OHTRZ*

Subject C a,-OHTRZ* 4-OHTRZ*

0.25-mg dose 6--12 12-18 18-24

45.4 4.4 4.1

2.6 0.5 0.9

26.7 4.4 1.8

1.2 0.6 0.3

25.1 7.3 2.7

1.8 1.3 0.4

0.5-mg dose 6--12 12-18 18-24 24-48

33.9 6.0 2.3 1.3

1.2 0.6 0.3 0.2

37.4 6.3 2.2 3.2

1.4 0.9 0.3 0.2

43.8 5.4 4.4 1.7

2.9 0.8 0.6 0.4

* Dataareexpressedasa percentageof the dose.

241


unchanged triazolam was monitored, but it was detected only in the first and second urine samples after a 0.5-rag ingestion (limit of detection: 0.5 ng/mL, data not shown). Therefore, unchanged triazolam was omitted in subsequent studies. As shown in Table I, the recovery percentages of (z-OHTRZ and 4-OHTRZranged between 91.2 and 97.6%. The calibration curves were linear in the range of 2-400 ng/mL for ~-OHTRZ and 1-25 ng/mL for 4-OHTRZ, with correlation coefficients that were routinely greater than 0.998. Samples that were found to contain a-OHTRZ in excess of the upper limits of linearity were reanalyzedafter dilution with negative urine. Table II shows accuracy and intra- and interassay precision data. The accuracy was within a 13.0% deviation from nominal values. The CVfor the intra- and interassay was within 10.4% at three concentrations for the two analytes.

munoresponses for unchanged triazolam and its metabolitesas a "nordiazepam equivalent". As a result, the displayed values may not accuratelyreflectthe concentrationof or In the present study,the human urinary excretionprofilesof a-OHTRZ and 4-OHTRZ were examined using LC-MS. Because this method has the capability of determining or and 4OHTRZseparately,it is superior to EMIT. Several commercial immunoassay systemsother than EMIT were also used in a preliminary screening of benzodiazepinesin urine. The mOHTRZconcentrations that gave positive results were reported to be as follows: 100-200 ng/mL for EMITd.a.u., 100-200 ng/mL for reformulated Microgenics CEDIA, 500 ng/mL for Abbott FPIA, and 5000 ng/mL for Bio Site TRIAGE (9,10). In the present study, the maximum concentrations of m/z 359

Excretion profiles of o~OHTRZ and 4-OHTRZ

o o.T.'z

8

The individual excretion patterns of (~-OHTRZ and 4-OHTRZ are shown in Figure 2. The metabolite concentrations in urine were proportional to the doses, and the excretion patterns of the two doses were similar. The maximum concentration of ~-OHTRZ was observed in the first or second samples obtained after ingestion (7.3-10.8 h after ingestion). However, the maximum concentration of 4-OHTRZ did not always parallel that for cz-OHTRZ. In several cases,the peaks corresponding to the two metabolites appeared in the range of 17.7-22 h after ingestion. Figure 3 shows the profiles of the o~-OHTRZI4-OHTRZratios. The ratios were initially more than 19.7 (range: ]9.7-52.9), then decreased rapidly, reaching a nearly constant value for times in excess of 12 h.

m/z 282

0

5

10

15

Figure 1. Typical ion chromatograms of a urine extract (11.5 h after the

ingestionof O.25-mgdoseof triazolam).

0.25-mg dose

0.5-mg d o s e 1000

i1 6

12 18 Time after ingestion (h)

~" 1000

24

or too1

~

9

e

c

o

9

"

0

o

"~

I

o~=~

9

%

1

9 12 18 Time after ingestion (h)

o

12

.

18 24 30 36 42 Time after ingestion (h)

] 9

48

subject B

0

100]

e

1~

oo o ~ 6

12

Oo

o" o

9 o

i

10

0

0 12 18 Time after ingestion (h)

a.. 18 24

~'1000

~ ]. ~" lOOt" "-

[o o

o9

9

o

9

o

0

30

36

42

48

Time after ingestion (h)

9 o

9

oeo"o .

1

24

subject C

8

9

v

oo

8

.

6 ~'1000

subject B 9

Discussion

242

1' 1"ooli ... oo

6

Knowledge of the excretion profile of triazolam metabolites is important because urine samples are not always obtained from victims immediately after the crimes are committed. Beck and colleagues reported on the human urinary excretionprofileof (~-OHTRZfor a clinical dosageof triazolam using an immunoassay system, an EMIT benzodiazepine assay (8). However, EMIT indicates the sum of the ira-

20

Retention time (rain)

Percent dose recovery of triazolam

metabolites The percent dose recoveries of a-OHTRZ and 4-OHTRZ from urine are shown in Table III. Most of the metabolites were excreted within the first 12 h. The total amount of metabolites excreted over the first 24 h was nearly proportional to the doses and no significant differenceswere noted in the overall excretion patterns between the two dosages. On average, 40.6%/3.2% for a 0.25-rag dose and 47.2%/3.0% for a 0.5-rag dose were excretedas a-OHTRZ/4-OHTRZ during the first 24 h.

4.o.T.z

24

1l 6

subject C

iiii:: ,

12

*9 9 Oo

o

18 24 30 36 42 Time after ingestion (h)

48

Figure 2. Urinaryexcretionprofilesof 0~-OHTRZ(o) and 4-OHTRZ(o) forsubjectsA, B, and C

after ingestionof triazolam (0.25 mg and 0.5 mg). Eachdatapoint representsthe meanof duplicatedeterminations.


30. 25,

0.25-mg dose

o

2o! 9 is. ,_\R

","b,.-.,

~I-- 10,

g s~ 0 12 18 Time after ingestion (h)

6

24

strates in women (12). According to Greenblatt et al. (13), a tendency for the weight-normalized clearance of triazolam was found to be higher in women than in men, although the difference was not statistically significant. Therefore, for the purposes of this study, we assume that no sex-related differences existed for the excretion profiles of triazolam metabolites. In conclusion, the urinary excretion patterns of two major triazolam metabolites in a clinical dosagerange are reported. To our knowledge,this is the first report that demonstrates differencesin the excretion of mOHTRZ and 4-OHTRZin healthy volunteers.

60

0.5-mg dose

50 40

References

30

o

20 10 0 6

12

18

24

30

36

42

48

Time after ingestion(h)

Figure3. mOHTRZ/4-OHTRZ ratiosfor individualsA (e), B (o), andC

(1~1)after ingestionof triazolam (0.25 mg and 0.5 mg).

~-OHTRZwere determined to be 160.2-513.1 ng/mL for a 0.25rag dose and 324.8--614.2 ng/mL for a 0.5-rag dose. Our results show that the EMITd.a.u., the reformulated MicrogenicsCEDIA, and the Abbott FPIAmay be capable of detecting mOHTRZ in the clinical dosage range. However,even the EMITd.a.u., which was the most sensitiveto r was positiveonly in the first or second urine samples after ingestion. Therefore, we conclude that immunoassay systems are not always appropriate for the screening of urinary triazolam metabolites in cases where a clinical dosage is involved. Eberts and colleagues (2) reported that glucuronide-conjugated ~-OHTRZ disappeared from the plasma more quickly than glucuronide-conjugated4-OHTRZ. In this study, urinary ~-OHTRZ concentrations tended to reach a maximum concentration and then to decrease more rapidly than 4-OHTRZ. These urinary excretion patterns of mOHTRZ and 4-OHTRZ would be expected to be similar to pharrnacokinetic patterns in plasma. Eberts and colleagues (2) reported that ~-OHTRZ and 4-OHTRZ are excreted in the urine, accounting for 53% and 8% over the first 24 h after an oral dose of 0.88 mg of 14C-triazolam. These data are higher than our data. This difference may be due to interracial differences in hepatic CYP3Aactivities between American and Japanese subjects. Midazolarn, a typical CYP3A4substrate, has been reported to show a lower hepatic clearance in Japanese subjects than in European or American subjects (11). However, such a conclusion cannot be made based on only this study. Victims of sexual assault under the influence of hypnotic drugs are generally female, and only male subjects participated in this study. The activity of CYP3Amight be higher in women than in men, leading to higher clearances of some CYP3Asub-

1. T. Kronbach, D. Mathys, M. Umeno, F.J. Gonzalez, and U.A. Meyer. Oxidation of midazolam and triazolam by human liver cytochrome P450111A4.Mol. Pharmacol. 36:89-96 (1989). 2. ES. Eberts, Y. Philopoulos, L.M. Reineke, and R.W. Vliek. Triazolam disposition. Clin. Pharmacol. Ther. 29" 81-93 (1981 ). 3. B. Marc, F. Baudry, P. Vaquero, L. Zerrouki, S. Hassnaoui, and H. Douceron. Sexual assault under benzodiazepine submission in a Paris suburb. Arch. Gynecol. Obstet. 263:193-197 (2000). 4. M.A. EISohly, S. Feng, S.J. Salamone, and R. Wu. A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine. J. Anal. Toxicol. 21:335-340 (1997). 5. A. Smith-Kielland, B. Skuterud, K.M. Olsen, and J. Morland. Urinary excretion of diazepam metabolites in healthy volunteers and drug users. Scand. J. Clin. Lab. Invest. 61:237-246 (2001). 6. W. Fishman, B. Springer, and B. Brunetti. Application of an improved glucuronidase assay method to the study of human blood [3-glucuronidase. J. BioL Chem. 173:449-456 (1948). 7. K. Tsujikawa, K. Kuwayama, T. Kanamori, Y. Iwata, Y. Ohmae, H. Inoue, and T. Kishi. Optimized conditions for the enzymatic hydrolysis of o~-hydroxytriazolam-glucuronide in human urine. J. Health 5ci. 50:286-289 (2004). 8. O. Beck, P. Lafolie, P. Hjemdahl, S. Borg, G. Odelius, and R Wirbing. Detection of benzodiazepine intake in therapeutic doses by immunoanalysis of urine: two techniques evaluated and modified for improved performance. Clin. Chem. 38:271-275 (1992). 9. A.D. Fraserand R. Meatherall. Comparative evaluation of five immunoassays for the analysis of alprazolam and triazolam metabolites in urine: effects of lowering the screening and GC-MS cut-off values. J. Anal Toxicol. 20:217-223 (1996). 10. A.D. Fraserand R. Meatherall. Improved cross-reactivity to alpha OH triazolam in the BMC CEDIA DAU urine benzodiazepine assay. Ther. Drug Monit. 20:331-334 (1998). 11. T. Tateishi, M. Watanabe, H. Nakura, M. Asoh, H. Shirai, Y. Mizorogi, S. Kobayashi, K.E. Thummel, and G.R. Wilkinson. CYP3A activity in European American and Japanese men using midazolam as an in vitro probe. Clin. Pharmacol. Ther. 69: 333-339 (2001). 12. J.C. Gorski, D.R. Jones, B.D. Haehner-Daniels, M.A. Hamman, E.M. O'Mara, and S.D. Hall. The contribution of intestinal and hepatic CYP3A4 to the interaction between midazolam and clarithromycin. Clin. PharmacoL Ther. 64:133-143 (1998). 13. D.J. Greenblatt, J.S. Harmatz, L.L.V. Moltke, C.E. Wright, A.L.B. Durol, L.M. HarreI-Joseph,and R.I. Shader.Comparative kinetics and response to the benzodiazepine agonists triazolam and zolpidem: evaluation of sex-dependent differences. J. PharmacoL Exp. Ther. 293:435-443 (2000). Manuscript accepted April 29, 2004; revision received September 7, 2004.

243