miyaguchi et al. - 2006 - a method for screening for various sedative-hypnotics in serum by liquid...

A method for screening for various sedative-hypnotics in serum

by liquid chromatography/single quadrupole mass spectrometry

Hajime Miyaguchi *, Kenji Kuwayama, Kenji Tsujikawa, Tatsuyuki Kanamori,Yuko T. Iwata, Hiroyuki Inoue, Tohru Kishi

National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa-shi, Chiba 277-0882, Japan

Received 4 June 2004; received in revised form 18 February 2005; accepted 7 March 2005

Available online 24 May 2005

www.elsevier.com/locate/forsciint

Forensic Science International 157 (2006) 57–70

Abstract

A screening method for the detection of sedative-hypnotics in serum is described. The target drugs, which include practically

all the sedative-hypnotics distributed in Japan, consisted of 5 barbiturates, 30 benzodiazepine-related drugs and 11 other sedative-

hypnotics (i.e., apronalide, bromisovalum, chloral hydrate, triclofos, chlorpromazine, promethazine, diphenhydramine, hydro-

xyzine, zopiclone, zolpidem and tandospirone). Thirty-nine analytes, selected in terms of the pharmacokinetics of the target drugs,

in human serum were screened using a combination of mixed-mode solid-phase extraction and liquid chromatography/

electrospray-ionization single-quadrupole mass spectrometry. The detection limits (non-basic analytes, 1–50 ng/ml; basic

analytes, 0.1–5 ng/ml) were sufficient to permit the screening of a single therapeutic administration of a target drug.

# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Drug screening; Benzodiazepines; Serum; Solid-phase extraction; Liquid chromatography/mass spectrometry (LC/MS)

1. Introduction

The use of sedative-hypnotics has been traced to various

crimes such as murder, sexual assault and traffic accidents.

In addition, these drugs have also been associated with

suicide by carbon monoxide poisoning and drug overdose.

Therefore, it is important to prepare a screening method for

all the sedative-hypnotics distributed in each country for

determining whether or not sedative-hypnotics are involved

in these cases.

Several immunoassay kits are available for screening

selected sedative-hypnotics, such as benzodiazepines and

barbiturates, but some have a limit of detection that may be

insufficient to reveal administration of a therapeutic dose [1].

* Corresponding author. Tel.: +81 4 7135 8001;

fax: +81 4 7133 9173.

E-mail address: [email protected] (H. Miyaguchi).

0379-0738/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights r

doi:10.1016/j.forsciint.2005.03.011

Capillary gas chromatography/mass spectrometry (GC/MS)

is the most reliable technique for drug identification due to

its excellent chromatographic resolution and the availability

of library-searchable spectral information using electron

ionization. As a result, a number of screening methods

for sedative-hypnotics that involve the use of GC/MS have

been reported [2,3]. However, some of the sedative-hypno-

tics have low volatility and require derivatization prior to

analysis. An additional ionization method (i.e., negative

chemical ionization) may be also required to determine

sub-ppb levels of sedative-hypnotics in blood [4].

Liquid chromatography/mass spectrometry (LC/MS) is an

alternative technique for drug identification. The high sensi-

tivity and wide applicability of LC/MS has led to dramatic

changes in drug analysis and a number of drug screening

methods using LC/MS(/MS) have been reported [5–9].

In particular, electrospray ionization provides outstanding

sensitivity for the detection of most sedative-hypnotics that

eserved.

H. Miyaguchi et al. / Forensic Science International 157 (2006) 57–7058

are charged, polar substances. While triple-quadrupole

LC/MS permits advanced sensitivity and selectivity, single-

quadrupole LC/MS has the merits of low cost, ease of

maintenance and adequate selectivity, all of which are suitable

for general screening [10].

Fractionating extraction is suitable for electrospray ioni-

zation because either the positive or negative mode must be

chosen along with the charge of the analytes. Mixed-mode

solid-phase extraction, the sorbent of which provides both

ion exchange and reversed phase properties, is a simple

technique that can be used for fractionation [1,6,11].

Both blood and urine are frequently used in drug testing.

In general, the total concentration of a drug and its meta-

bolites in urine is higher than that in blood, but urine would

require the hydrolysis of a conjugate followed by analyzing

phase I metabolites, standards for which are not always

commercially available. Using blood for drug screening

would avoid most of the problems mentioned above, but

a higher sensitivity would be required in order to detect

smaller amounts of compounds.

The goal of this study was to establish a screening method

for sedative-hypnotics use. The target drugs included all the

hypnotics that have been approved by the Japanese govern-

ment for the treatment of insomnia or sleep disorders as of

2004 and included barbiturates, benzodiazepine-related com-

pounds as well as other drug categories [12,13] (Table 1). In

addition, the benzodiazepines for the treatment of anxiety and

epilepsy (e.g., diazepam and clonazepam) and some sedatives

rather than hypnotics (e.g., hydroxyzine) were also included.

Herbal medicines were excluded from the list of targets

because of their low potency. The present method was devel-

oped on serum samples, and 39 analytes were selected in terms

of their pharmacokinetics. A mixed-mode solid-phase extrac-

tion and a single-quadrupole LC/MS was used for extraction

and detection, respectively.

2. Experimental

2.1. Reagents

Methanol, acetonitrile (both HPLC grade), 25% aqueous

ammonia, ammonium acetate and ethenzamide (internal

standard) were purchased from Wako (Osaka, Japan). Fol-

lowing human sera (a–f) were used: a (male, pooled), Lot

No. 122K0424, PN H1388, Sigma (St. Louis, MO, USA); b(male, blood type AB, pooled), Lot No. 083K0477, PN

H1513, Sigma; c (pooled), Lot No. R12393, PN 823201,

ICN (Irvine, CA, USA); d (individual, male, narcotics free),

Lot No. N57743, Cosmo Bio (Tokyo, Japan); e (individual,

male), Lot No. N59708, Cosmo Bio; f (individual, female),

Lot No. N59732, Cosmo Bio. Serum a was used for the

blank serum unless otherwise described. Water was purified

and filtered through a Milli-Q Simpli Lab-UV system from

Millipore (Billerica, MA, USA). Other chemicals were of

analytical reagent grade.

2.2. Standards and solutions

The target drugs and the corresponding analytes

are shown in Table 1. An analytical standard of 7-bromo-

5-(2-fluorophenyl)-3H-1,4-benzodiazepine-2(1H)-one, an

active metabolite of haloxazolam (Sankyo code, No. 574;

see Fig. 1d), was synthesized from the corresponding

benzophenone (prepared by the hydrolysis of haloxazolam)

and glycine ethyl ester as described in the literature [14].

Amobarbital (Nippon Shinyaku), flutazolam (Mitsui phar-

maceutical), lormetazepam (Wyeth), quazepam (SS phar-

maceutical), rilmazafone M-4 (see Fig. 1f, Shionogi),

fludiazepam and tandospirone citrate (both Sumitomo phar-

maceuticals) were generously donated by corresponding

pharmaceutical companies. Bromisovalum, phenobarbital,

trichloroacetic acid, brotizolam, clotiazepam, etizolam,

nimetazepam, nitrazepam and promethazine were pur-

chased from Wako. Other standard compounds were pur-

chased from Sigma. Stock solutions (10 mg/ml) of non-

basic analytes (A, see Table 1) were prepared in acetoni-

trile:water (7:3, v/v). Stock solutions (1 mg/ml) of basic

compounds (B, see Table 1) were prepared in acetonitrile

except for zolpidem (1 mg/ml in acetonitrile:water, 7:3, v/

v) and rilmazafone M-4 (0.5 mg/ml in acetonitrile:

water, 7:3, v/v). Working mixtures of analytes were pre-

pared from the respective stock solutions of A and B. All

stock solutions and working mixtures were kept at 4 8C and

no degradation of the compounds was observed in the

working mixture (A, 50 mg/ml; B, 5 mg/ml, in water:ace-

tonitrile, 3:1, v/v) by LC/UV measurements after 2 months

of preparation. A 10-mmol/l ammonium acetate solution

(pH 6.8), for the mobile phase, was prepared daily by the

500-fold dilution of an ammonium acetate stock solution

(5 mol/l, filtrated) with fresh Milli-Q water with no sub-

sequent filtration.

2.3. Instrumentation

The LC/MS equipment was composed of a 2690 separa-

tion module, a ZQ single-quadrupole mass detector and Mass

Lynx software (Waters, Milford, MA, USA). A Waters 996

photodiode array detector was used for UV measurements.

The chromatographic conditions were based on a LC/MS

screening method developed by Adachi and Takahashi [15].

A Symmetry C18 column (Waters, 150 mm � 2.1 mm, par-

ticle size 3.5 mm) was used for the separation at 35 8C with

an Opti-solv in-line filter (0.5-mm pore, Optimize technol-

ogies, OR, USA). The mobile phase, delivered at a flow rate

of 0.2 ml/min, was a gradient of methanol (B) in 10 mmol/l

ammonium acetate (A): 0–5 min, 10% B; 5–45 min, from

10% to 90% linear gradient of B in A; 45–55 min, 90% B;

55–60 min, from 90% to 10% B; 60–80 min, equilibration of

the column with 10% B.

Electrospray ionization was performed with a Z-spray

module (Waters) with no splitting from the column. Acqui-

sition programs were constructed for each fraction, and the

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9Table 1

The analytes and acquisition parameters

Type Analyte Related sedative/hypnotics Molecular

formula

Mono-isotopic

massa

Monitored ions ( m/z )b Typical

retention

time (min)

Acquisition period

(min)c

Cone voltage (V)d

A Amobarbital C11H18N2O3 226 225 32.8 28–40 �30

A Apronalide C9H16N2O2 184 185 30.2 28–40 30

A Barbital C8H12N2O3 184 183 16.5 10–20 �30

A Bromisovalum C6H11BrN2O2 222, 224 223, 225, 180, 182 25.6 20–28 30, 60

A Pentobarbital C11H18N2O3 226 225 32.8 28–40 �30

A Phenobarbital C12H12N2O3 232 231 24.9 20–28 �30

A Secobarbital C12H18N2O3 238 237 34.6 28–40 �30

A Trichloroacetic acid (TCA) Triclofos, chloral hydrate C2HCl3O2 162 161, 163, 117, 119 5.3 2.5–10 �20, �30

B Alprazolam C17H13ClN4 308 309, 311 35.2 34.5–36.25 60

B Bromazepam C14H10BrN3O 315, 317 316, 318 31.3 28–34.5 50

B Brotizolam C15H10BrClN4S 392, 394 393, 395 35.7 34.5–36.25 50

B Chlordiazepoxide C16H14ClN3O 299 300 37.2 36.25–37.75 40

B Chlorpromazine C17H19ClN2S 318 319, 321 44.5 39.5–50 45

B Clonazepam C15H10ClN3O3 315 316, 318 33.0 28–34.5 50

B Clotiazepam C16H15ClN2OS 318 319, 321 40.4 39.5–50 45

B Delorazepam Cloxazolam, mexazolam C15H10Cl2N2O 304 305, 307 37.0 36.25–37.75 60

B N-Desmethyldiazepam Chlordiazepoxide, clorazepate,

diazepam, medazepam, oxazolam,

prazepam

C15H11ClN2O 270 271, 273 37.7 37.4–38.5 60

B N-Desmethylfludiazepam Ethyl loflazepate, fludiazepam,

flurazepam, flutazolam,

flutoprazepam, quazepam

C15H10ClFN2O 288 289 36.0 34.5–36.75 50

B Diazepam Medazepam C16H13ClN2O 284 285, 193 38.5 37.75–39.5 60, 90

B Diphenhydramine C17H21NO 255 256 35.3 34.5–36.75 20

B Estazolam C16H11ClN4 294 295 34.0 28–34.5 50

B Etizolam C17H15ClN4S 342 343 36.2 34.5–36.75 50

B Fludiazepam C16H12ClFN2O 302 303, 305 37.2 36.25–37.75 60

B Flunitrazepam C16H12FN3O3 313 314 33.3 28–34.5 50

B Flutazolam C19H18ClFN2O3 376 377 38.0 37.4–38.5 60

B Haloxazolam No. 574 Haloxazolam C15H10BrFN2O 332, 334 333, 335 36.7 36.25–37.75 60, 40

B Hydroxyzine C21H27ClN2O2 374 375 43.1 39.5–50 50

B Lorazepam Cloxazolam, delorazepam,

lormetazepam, mexazolam

C15H10Cl2N2O2 320 321 35.3 34.5–36.25 45

B Lormetazepam C16H12Cl2N2O2 334 335, 337 36.6 36.25–37.75 40

B Midazolam C18H13ClFN3 325 326 38.5 37.75–39.5 60

B Nimetazepam C16H13N3O3 295 296 33.7 28–34.5 50

B Nitrazepam Nimetazepam C15H11N3O3 281 282 32.8 28–34.5 50

B Promethazine C17H20N2S 284 285 41.8 39.5–50 30

B Quazepam C17H11ClF4N2S 386 387 41.7 39.5–50 50

H. Miyaguchi et al. / Forensic Science International 157 (2006) 57–7060le

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parameters (monitored ions, acquisition timetable and cone

voltage) are shown in Table 1. Other conditions were as

follows: dwell time, 0.1 s; capillary voltage, 3 kV; extractor,

3 V; rf lens, 0 V; source temperature, 100 8C; desolvation

temperature, 300 8C; cone gas (nitrogen) flow, 50 l/h; des-

olvation gas (nitrogen) flow, 350 l/h.

When examining the optimum cone voltage, an analy-

tical standard solution was directly introduced into the mass

spectrometer and a series of cone voltages (from 10 V to

70 V, 10 V intervals) was applied to the scan acquisition for

each standard analyte.

2.4. Adsorption of the analytes on membrane filters

The analytes were divided into six groups in order to

avoid peak overlapping in the chromatogram, and 100 ml of

the mixture (A, 10 mg/ml; B, 1 mg/ml) in water:methanol

(9:1 or 1:9, v/v) was filtered through an Ultrafree MC (a

centrifugal filter unit with a 0.45-mm polyvinylidene fluoride

(PVDF) microporous membrane, Nihon Millipore, Yone-

zawa, Japan). Differences in peak areas of analytes after

filtration were monitored by LC/UV or LC/MS measure-

ments (n = 2). The monitored wavelength and monitored

ions are shown in Table 2.

2.5. Sample preparation and extraction procedure

A 0.5-ml volume of human serum (spiked with appro-

priate amounts of the standard mixtures) was mixed with

0.5 ml of 0.1 mol/l HCl. After brief mixing, the solution was

applied to an Oasis MCX mixed-mode solid-phase extrac-

tion cartridge (Waters, 1-ml syringe volume, 30-mg sorbent

weight) conditioned with 1 ml of acetonitrile and 2 ml of

water. The cartridge was subsequently washed with 0.5 ml of

0.1 mol/l HCl. After drying under vacuum for 30 s, the first

elution was carried out with 1 ml of acetonitrile. The car-

tridge was dried again under vacuum for 30 s, followed by a

second elution with 1 ml of acetonitrile–25% aqueous

ammonia (19:1, v/v). After 10 ml of glycerol:water (1:1,

v/v) was added to each fraction, the eluates were evaporated

separately under a gentle stream of nitrogen at 45 8C, giving

glycerol droplets. The residues were redissolved in 50 ml of

water:methanol (9:1 for the first fraction, 1:9 for the second

fraction, v/v), followed by membrane filtration through

Ultrafree MC filters. The filtrates were transferred to plastic

vials, and a pair of the extracts was subject to the LC/MS

analyses separately using the corresponding acquisition

programs. Twenty microliters of each was injected via the

built-in autosampler.

2.6. Extraction recovery of Oasis MCX cartridge

Drug concentrations for recovery tests were 500 ng/ml

(A) and 50 ng/ml (B) in serum (n = 5). In order to avoid peak

overlapping especially between amobarbital and pentobar-

bital, the analytes were divided into two groups and

H. Miyaguchi et al. / Forensic Science International 157 (2006) 57–70 61

Table 2

Filter adsorption, extraction recoveries and detection limits

Type Analyte Filtration recoveries (%, n = 2) Extraction

recoveries

(%, n = 5)

Detection limits (ng/ml)

Monitor

wavelength

(nm)

Dissolved

in 10%

methanol

Dissolved

in 90%

methanol

Recovery S.D. Serum d Serum e Serum f

A Amobarbital 216 94.9 98.3 100.5 4.8 1 1 5

A Apronalide 240 97.2 98.8 99.5 6.1 1 1 5

A Barbital 213 96.2 98.4 99.0 6.4 10 10 10

A Bromisovalum 232 99.6 – 101.2 4.8 10 10 10

A Pentobarbital 216 99.6 99.1 87.4 4.1 1 1 5

A Phenobarbital 240 99.5 – 92.4 7.0 5 5 5

A Secobarbital 216 94.5 98.0 102.2 4.9 1 1 5

A Trichloroacetic acid 211 99.3 – 81.3 2.4 50 50 50

B Alprazolam 226 97.2 100.3 91.5 2.0 0.5 0.5 0.5

B Bromazepam 237 98.0 101.5 97.7 1.6 1 1 1

B Brotizolam 243 95.3 94.7 96.0 3.2 0.5 0.5 0.5

B Chlordiazepoxide 263 80.4 99.3 107.7 3.0 1 1 0.5

B Chlorpromazine 256 7.6 36.2 82.3 2.4 1 1 1

B Clonazepam 308 91.6 104.4 80.4 4.9 1 1 1

B Clotiazepam 244 79.3 94.6 86.6 2.1 0.1 0.5 0.1

B Delorazepam 232 93.0 100.2 97.1 6.6 0.5 0.5 0.5

B N-Desmethyldiazepam 232 85.7 103.8 87.0 4.3 0.5 0.5 0.5

B N-Desmethylfludiazepam 232 89.3 98.8 96.6 3.1 0.1 0.1 0.1

B Diazepam 232 85.4 99.7 96.6 4.3 0.1 0.1 0.1

B Diphenhydramine m/z 256 (MS) 19.2 78.5 83.2 2.9 0.5 0.5 0.5

B Estazolam 232 97.2 100.3 93.5 1.6 0.5 0.5 0.5

B Etizolam 244 89.0 103.4 91.2 7.7 0.1 0.1 0.1

B Fludiazepam 234 90.3 96.2 90.8 3.4 0.1 0.1 0.1

B Flunitrazepam 308 85.3 98.0 94.3 3.6 0.5 0.5 0.5

B Flutazolam 246 62.6 101.9 83.8 3.1 1 1 1

B Haloxazolam No. 574 232 93.0 99.1 88.0 6.5 0.5 0.5 0.5

B Hydroxyzine m/z 375 (MS) 6.4 94.1 93.4 1.2 1 1 1

B Lorazepam 232 96.9 100.0 81.9 4.2 5 5 5

B Lormetazepam 233 93.8 98.6 45.0 13.4 5 5 5

B Midazolam 232 72.9 98.2 83.3 3.5 0.1 0.1 0.1

B Nimetazepam 262 91.5 95.0 86.3 5.0 0.5 0.5 0.1

B Nitrazepam 260 96.3 100.3 89.1 2.8 0.5 0.5 0.5

B Promethazine 253 2.7 78.6 94.6 3.6 1 1 1

B Quazepam 287 23.7 100.0 61.1 25.3 5 5 5

B Rilmazafone M-4 234 100.8 97.0 36.9 6.4 1 1 1

B Tandospirone 244 73.8 77.1 91.1 2.8 0.1 0.1 0.1

B Triazolam 227 92.0 102.2 90.6 4.5 0.5 0.5 0.5

B Zolpidem 244 61.0 90.5 87.9 3.7 0.1 0.1 0.1

B Zopiclone 305 73.1 43.4 90.0 2.1 5 5 5

–, not determined due to peak distortion; S.D., standard deviation.

investigated by LC/MS separately. To compensate for the

volume variation of the final LC/MS samples, each extract

was dissolved in 50 ml of water:methanol (9:1 for the first

fraction, 1:9 for the second fraction, v/v) containing 250 ng/

ml ethenzamide and the calculations were performed by the

peak–area ratios to ethenzamide. The monitoring ion and

cone voltage for ethenzamide were m/z 166 V and 30 V,

respectively, and the retention time of ethenzamide almost

corresponded to that of phenobarbital. When preparing

reference samples, one of the two standard mixtures

was added to each fraction of a blank serum extract, and

the average (n = 3) was considered as 100% value of the

recovery.

2.7. Detection limits

The following concentrations (n = 2) were employed for

determining the detection limits: A, 50, 10, 5, 1 (ng/ml);


Fig. 1. Metabolic pathways for sedative-hypnotic drugs in circulating blood.

B, 5, 1, 0.5, 0.1 (ng/ml). Analytes were divided into two

groups as mentioned above. Three human sera (d–f) were

used as matrices. The concentration required to produce a

signal-to-noise ratio of better than 5:1 (determined by the

peak-to-peak method) was accepted as the detection limits.

2.8. Matrix effects

Six human sera (a–f), obtained commercially, were

submitted to extraction as described above to evaluate the

existence of the endogenous interferences that may cause


Fig. 2. Infusion experiment. For the blank run, water:methanol (9:1, v/v) was injected into the column instead of a serum extract. Cone voltages

used for the monitoring at m/z 117 and 163 were 30 V and 20 V, respectively. RT, retention time; TCA, trichloroacetic acid.

false positive. By using the same extracts of sera d–f,postcolumn infusion experiments [16] were carried out.

Illustration of the experiments is shown in Fig. 2 (upper).

Chromatographic separations for the serum extracts were

carried out while trichloroacetic acid (TCA, 20 mg/ml) in

water:methanol (1:1, v/v) was continuously infused post-

column through a tee at a flow rate of 2 ml/min. At the same

time, negative ESI signals of TCA (scan mode, cone voltage

�20 V and �30 V) were observed.

3. Results

3.1. LC/MS conditions

Mass chromatograms of the extracts obtained from the

spiked serum are shown in Fig. 3. Satisfactory separation was

achieved for most of the analytes by employing relatively long

gradient cycle (80 min), although careful choices of the

monitored ions were necessary for resolving overlapped

peaks, as described below. Stability of the retention times

was generally excellent, but retention times of diphenhydra-

mine, chlorpromazine and promethazinewere liable to shift by

the concentration of the analytes and condition of the column.

For the maximum MS response, the optimum polarity

and cone voltages were examined for each analyte, and these

values were employed for the settings of selected ion

monitoring (SIM), along with the fractionation and retention

time of the analytes (Table 1).

Some minor isotopes were monitored to resolve over-

lapping peaks. For example, the second isotopic peak at m/z

311 in addition to the first at m/z 309 was employed for

the detection of alprazolam because the peak at m/z 309

overlapped with that for zolpidem (Fig. 3). Similarly, the


Fig. 3. Mass chromatograms of serum extracts. Serum d was used for the matrix. Non-basic analytes (A (except for amobarbital), 1000 ng/ml in

serum) and basic analytes (B, 100 ng/ml in serum) were extracted in the first fraction (upper) and the second fraction (lower), respectively. RT,

retention time; n, negative mode; TCA, trichloroacetic acid.


Fig. 4. Mass spectra of lormetazepam and haloxazolam metabolite

No. 574. Cone voltages were set to 30 V (lormetazepam) and 50 V

(the haloxazolam metabolite).

retention time and the base mass peak for lormetazepam

were consistent with those of the haloxazolam metabolite

(Fig. 3), but it was possible to resolve them by comparing the

spectral patterns (lormetazepam, m/z 335 and 337; the

haloxazolam metabolite, m/z 333, 335 and a trace of

337). The full-scan MS spectra of these two compounds

are shown in Fig. 4.

Amobarbital could not be discriminated from pentobar-

bital by this method because the retention times and mass

spectra were completely identical.

Fig. 5. Comparison of the mass chromatograms of bromisov

3.2. Sample preparation

In order to minimize loss of analytes by evaporation,

glycerol was added prior to the evaporation process. The

addition of glycerol improved the recovery of TCA; never-

theless, excess evaporation impaired the recovery (data not

shown).

Adsorption losses of the analytes on PVDF membranes

were evaluated (Table 2). An extract is usually reconstituted

in the initial mobile phase, but dissolving in 10% methanol,

equal to the initial mobile phase of this method, resulted in

the considerable losses of some basic analytes such as

chlorpromazine, diphenhydramine, hydroxyzine, prometha-

zine and quazepam on filtration. Dissolving the second

fraction (B) in 90% methanol, which had no effect on

retention times and peak shapes, dramatically improved

the adsorption losses of hydrophobic analytes on filtration.

Although recovery of chlorpromazine was still low (36.2%)

and recovery of zopiclone was getting low (43.4%), water:

methanol (1:9) was employed for the reconstitution of the

second fraction.

Extraction efficiencies are shown in Table 2. All of the

analytes were extracted in the expected order and the

extraction efficiencies were sufficient for screening although

some of the recoveries were relatively low (i.e., lormetaze-

alum extracted from different sera. RT, retention time.


pam, quazepam and rilmazafone M-4). Most benzodiaze-

pines were eluted into the second fraction only, but lorme-

tazepam was eluted into the first fraction as well as the

second fraction (data not shown).

Fig. 6. Mass chromatograms derived from a real-case sample. The order of

bromisovalum, apronalide and diphenhydramine, respectively.

3.3. Detection limits

Detection limits in serum were determined (Table 2).

Three sera, derived from different donors, were used for the

chromatograms are consistent with Fig. 3. Peaks #1–3 correspond to


matrices. The non-basic analytes were detectable at con-

centrations above 1–10 ng/ml in serum except for TCA

(50 ng/ml), and the basic analytes were detectable at con-

centrations above 0.1–1 ng/ml in serum except for loraze-

pam, lormetazepam, quazepam and zopiclone (5 ng/ml).

There was substantially no difference in the detection limits

between sera.

3.4. Matrix effects

By monitoring isotopic ions and fragment ions in addi-

tion to the major protonated molecules, interferences were

discriminated from the analytes. Examples of bromisovalum

(50 ng/ml in sera d–f) are shown in Fig. 5. The retention time

of bromisovalum was 26.0 min. The peaks at m/z 223 and

225 are derived from the protonated molecules, and the

peaks at m/z 180 and 182 are derived from the fragment ions

of bromisovalum. Large interferences were observed

near the peaks of bromisovalum at m/z 223 and 182; on

the other hand, the peaks of bromisovalum at m/z 225 and

180 were free from interference. Any interference that was

unable to discriminate from the analytes (at concentrations

higher than the detection limits) was not observed in six

blank matrices.

The postcolumn infusion experiment resulted in Fig. 2.

Severe ion suppression and interferences were observed in

the early period of the chromatography cycle, but at the

retention time of TCA, no matrix effect that would impair

the signal intensity was observed. Similarly, barbital (ESI

negative, cone voltage �30 V) and bromisovalum (ESI

positive, cone voltage 30 V and 60 V) were tested, and no

suppression was observed at the retention time of the

corresponding drug (data not shown).

3.5. Forensic application

The present method was applied to a case of suicide by

burning charcoal in a small space. Empty packages of non-

prescription medicines containing bromisovalum, aprona-

lide and/or diphenhydramine were found at the scene. This

screening resulted in the detection of these three compounds

(peaks #1–3 in Fig. 6), and the result was verified by GC/MS

analyses (data not shown).

4. Discussion

4.1. Choice of analytes based on pharmacokinetics

In general, drugs persist in circulating blood for at least

several hours after an oral administration, but some types of

sedative-hypnotics are quickly eliminated from the blood.

Therefore, in order to demonstrate the use of the target drugs,

analytes were chosen on the basis of pharmacokinetic data.

The metabolism of sedative-hypnotic drugs is summarized

in Table 1 and Fig. 1.

Chloral hydrate and triclofos are immediately biotrans-

formed to 2,2,2-trichloroethanol, the active metabolite, fol-

lowed by oxidation to TCA (Fig. 1a). It has been reported

that chloral hydrate is not detectable in plasma after a

therapeutic dose, but that TCA levels are higher than

5 mg/ml, 100-fold higher than the detection limit of this

method, even after 192 h [17]. A similar mechanism has

been proposed for the metabolism of triclofos [18]. There-

fore, TCA would be the most suitable alternative for

the demonstration of the two chlorinated drugs after their

ingestion (Table 1), although TCA is pharmacologically

inactive.

Some benzodiazepine derivatives contain metabolically

unstable moieties that are easily removed. Oxazolam, clox-

azolam, haloxazolam, mexazolam and flutazolam are ben-

zodiazepine-related compounds that have oxazole rings,

which are transformed to the corresponding benzodiazepines

(Fig. 1b–e). It has been reported that the parent drugs in

blood are undetectable (cloxazolam [19], haloxazolam [20]

and mexazolam [21]) or present in trace amounts in some

individuals (oxazolam [22]). Therefore, not the precursors

but the parent benzodiazepines were employed as the target

analyte (Table 1). In the case of flutazolam, it has been

reported that the parent drug, as well as both the correspond-

ing benzodiazepine and its N-desalkyl form (N-desmethyl-

fludiazepam), can be observed in serum, and the level of N-

desmethylfludiazepam in serum remained nearly constant

(about 5 ng/ml) for a day, whereas those of the parent drug

and the corresponding benzodiazepine decreased over time

[23]. Thus, monitoring of both the parent drug and N-

desmethylfludiazepam is preferable in terms of demonstrat-

ing flutazolam use.

Rilmazafone is a ring-opened benzodiazepine-derivative

that is a precursor of several active metabolites. After the

degradation of the glycine moiety of rilmazafone, it sponta-

neously forms a ring-closed benzodiazepine, followed by

demethylation and oxidation of the lateral N-dimethylamide

group [24] (Fig. 1f). It has been reported that the resulting

carbonate (M-4) is inactive but is the major metabolite in

plasma for periods of up to 6 h, while the parent compound

was not observed after therapeutic dosing [24]. Although an

analytical standard of rilmazafone M-4 is not available

commercially, the complete hydrolysis of rilmazafone by

refluxing in 3 mol/l HCl gave M-4 as a white precipitate

(data not shown).

The 1,4-benzodiazepines that have halogen atoms at

certain positions (i.e., 7-Cl and 20-F: ethyl loflazepate,

fludiazepam, flurazepam, flutazolam, flutoprazepam and

quazepam) are all metabolized to N-desmethylfludiazepam,

an active metabolite (Fig. 1b). Ethyl loflazepate, flurazepam,

flutazolam and flutoprazepam are immediately metabolized

to N-desmethylfludiazepam [23,25–27]. On the other hand, a

certain amount of fludiazepam and quazepam appears in

circulating blood after the administration of a therapeutic

dose [28,29]. Therefore, N-desmethylfludiazepam, fludiaze-

pam and quazepam were chosen for analytes.


N-Desmethyldiazepam (nordiazepam, nordazepam) is

also a common metabolite of certain benzodiazepines such

as diazepam [30], prazepam [30,31], oxazolam [22,31],

clorazepate [30,31], medazepam (via diazepam) [32] and

chlordiazepoxide [33] (Fig. 1c). Medazepam was not mon-

itored in this method, since it has been reported that

N-desmethyldiazepam is maintained at a higher level

(ca. 50–100 ng/ml) for several days after the oral adminis-

tration of medazepam, while the parent drug was observed

only in the early period and the level of diazepam was

maintained at a lower constant value (ca. 10 ng/ml),

although considerable differences between individuals were

noted [32]. The appearance of the other drugs in circulating

blood, not mentioned above, was confirmed by pharmaco-

kinetics data found in the literatures and package inserts of

the corresponding pharmaceutical products.

The detection limits for the analytes were acceptable,

although those of lorazepam, lormetazepam, quazepam and

zopiclone (5 ng/ml) were inferior to other basic analytes.

Lormetazepam has the lowest maximum blood concentra-

tion (Cmax) (12.4 ng/ml) among these four drugs, but elim-

ination of lormetazepam from circulating blood is relatively

slow (half life, 13.6 h) [34]. Therefore, the detection limit

estimated for lormetazepam is sufficient to demonstrate its

intake for about half a day after ingestion.

Because the present method focuses on screening, addi-

tional analyses such as GC/MS and LC/MS/MS are thought

to be essential to achieve definite identifications. Moreover,

because some of the analytes can be derived from two or

more drugs, the additional analyses are also required to

specify the ingested drug. It should be kept in mind that a

parent drug itself, such as diazepam, lorazepam, nitrazepam,

pentobarbital and phenobarbital, could be a metabolite of

others, as shown in Fig. 1 [32,34–37].

4.2. LC/MS conditions

In comparison with tandem LC/MS/MS measurements, a

single LC/MS measurement requires finer chromatographic

separation to discriminate possible drugs from a large

number of compounds that consist, not only of analytes,

but also of numerous interferences in biological matrices as

well, that is, high-throughput chromatography may not be

appropriate when single LC/MS is employed.

SIM enables a cone voltage at the optimum value to be set

which would provide the best sensitivity for each analyte,

and of course, SIM is essentially more sensitive than full-

scan mode in a quadrupole mass analyzer. On the other hand,

spectral information is not available with SIM. To compen-

sate the drawback of SIM, some minor isotopic ions and

some fragment ions were also monitored in this method,

which was useful for the discrimination of the analytes from

interferences (Fig. 5). Fragmentation was introduced only

for some interference-rich analytes because an increase in

the number of simultaneous acquisitions decreases the

number of data points per peak.

4.3. Sample preparation

Sample filtration prior to injection is important because

presence of particulates in the samples can reduce column

life. On the other hand, there is a possibility of adsorption on

the filter membrane. Using a methanol-rich solvent effec-

tively reduced adsorption of the hydrophobic analytes, such

as promethazine and hydroxyzine, on the fluoropolymer

filter membrane, although the recovery of zopiclone was

unexpectedly deteriorated.

The first fraction contained more endogenous interfer-

ences than the second fraction for two reasons: there is

considerable non-basic interference, and only aqueous wash-

ing was carried out before the first elution. A further washing

step was not employed because barbital and TCAwere eluted,

even with 5% methanol (data not shown). However, this is not

a serious problem because therapeutic levels of non-basic

sedative-hypnotics in blood are higher than those of basic

drugs, and ion suppression effects caused by the interferences

in the first fraction had been estimated in this study.

4.4. Matrix effects

Ion suppression caused by coeluted compounds is an

inevitable phenomenon for electrospray ionization. This can

deteriorate the response of the spectrometer, and conse-

quently, the detection limits. In the present method, polar

analytes that are barely retained by the analytical column,

such as TCA, are obviously at the high risk of ion suppres-

sion. Accordingly, infusion experiments of TCA, barbital

and bromisovalum were carried out for evaluating the

interferences that cause ion suppression. Moreover, the

detection limits of all the analytes were determined using

three individual sera as the matrices. These experiments

demonstrated that there is no evidence of ion suppression.

In conclusion, the present method will be useful for

preliminary screening of sedative-hypnotics drugs in serum.

From practical point of view, it will be beneficial to include

other drugs that cause sedation such as g-hydroxybutyrate,

classic antidepressants, reserpine and opiates. Further studies

on the applicability to real-case samples are now under

investigation.

Acknowledgements

We are grateful to Dr. Hitoshi Sekine (Saitama prefectural

police H.Q.) for providing drug standards. We also thank Dr.

Shinichi Suzuki (National Research Institute of Police

Science) for valuable information on drug metabolism.

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