Comparison of HPLC and GC-MS Methods for Determination of ...

Journal of Analytical Toxicology, Vol. 25, July/August 2001

Comparison of HPLC and GC-MS Methods for Determination of Embutramide (A Component of Tanax| or T-61| in Biological Specimens M. Giorgi 1, S. Bertini 2, G. Soldani1, and M. GiusianP,* 1Section of Pharmacology and Toxicology, Department of Veterinary Clinics, University of Pisa, Vie delle Piagge 2, 56124 Pisa, Italy; 2Institute of Food Inspection, University of Parma, V. del Taglio 8, 43100, Parma, Italy; and 3Section of Forensic Medicine and Toxicology, Department of Public Health, University of Pisa, V. Roma 55, 56100 Pisa, Italy

Abstract Tanax or T-61, a euthanasia solution commonly used in veterinary medicine, has been often involved in suicide attempts (humans) and malicious intoxications (animals). For forensic reasons, the identification of one or more of the three components (embutramide, mebenzonium iodide, and tetracaine hydrochloride) of Tanax is needed to confirm the hypothesis of intoxication. This study was performed with new high-performance liquid chromatographic and gas chromatographic-mass spectrometric methods to identify embutramide in biological matrices (blood, liver, kidney) from different animal species. The good sensitivity and specificity of both methods recommend their use in toxicological analysis in both human and veterinary medicine.

collapse because of paralysis of the intercostal muscles and diaphragm (6). Tetracaine hydrochloride belongs to the same group as procaine, but it is 20 times more potent; it is added to the solution because of its local ester anesthetic activity,which reduces painful tissue reactions at the injection site (7). DMF is an organic solvent commonly used in industry (e.g., in the manufacture and processing of plastics) (8). Tanax has often been involvedin deaths and suicide attempts of human beings and may also be a factor to be considered in cases of malicious death in apparently healthy animals such as race horses or dogs (9). Analytical determination of Tanax in human or animal tissues or biological fluids can support medical diagnosis. Since 1983, several analytical methods have

r

Introduction Tanax (T-61) is a euthanasia solution marketed since 1963 in Germany and available in Canada and in the rest of Europe (1--4); it is commonly used in veterinary medicine for its narcotic and curariformlike activity.This drug consists of a mixture of three active compounds: N-2-ethyl-2-(3-methoxyphenyl)butyl-4-hydroxybutanamide (embutramide, EMB) (200 mg/mL), 4,4'-methylene bis-N,N,N-trimethylcyclohexanaminium di-iodide (mebenzonium iodide) (50 mg/mL), and p-butylaminobenzoyl-dimethylamino-ethanol chloride (tetracaine hydrochloride) (5 mg/mL) (Figure 1). These compounds are dissolvedin a mixture ofN,N-dimethyl4ormamide(DMF) and water (6:4, v/v). EMB is a general anesthetic derived from gamma-hydroxybutyrate that possesses a strong narcotic effect and induces a deep anesthesia by paralyzing the brain centers that control breathing in the central nervous system (CNS) (5). Mebenzonium iodide exerts a curariformlike action, paralyzing the skeletal muscles and rapidly inducing a respiratory

~2"

"OCH3

N-2-Ethyl-2-(3-methoxyphenyl)-butyl-4-hydroxybuta namlde Embutramide (EMB) 81

CH3 ~

~

CH3

Ie

4,4'-Methylenebis-N,N, N-trimet~ylcyclohexanaminlumdlodide Mebenzonium iodide O

CH3 o/~N'-cH

3

H3C-A,J"~ p-Butylamlnobonzoyl-dlmethylamlno-ethanol chloride

Tetracaine hydrochloride Figure 1. Molecular formula of the three coformulants of Tanax.

"Authorto whomcorrespondenceshouldbe addressed.

Reproduction (photocopying)of editorial content of this journal is prohibited without publisher'spermission.

323


been employed to detect Tanax components, despite some difficulties: it is not easy to identify mebenzonium iodide in biological materials because of the low solubility of quaternary ammonium compounds in most of the common organic solvents. Furthermore, tetracaine hydrochloride is quickly degraded by esterase enzymes. EMB is the most widely investigated coformulation constituent. Different analytical methods to detect one or more constituents of Tanax have been pointed out and described in the literature: thin-layer chromatography followedby a quantitative estimation by UV-spectrophotometry (10), gas chromatography-mass spectrometry (GC-MS) equipped with electron-ionization at 70 eV (11,12), gas chromatography with nitrogen-phosphorus detection (13), and GC-MS after derivatization (14). The purpose of this study was to compare high-performance liquid chromatography (HPLC) and GC-MS methods for determination of EMB, a component of Tanax or T-61 in biological specimens.

v/v) adjusted to pH 4.7 with o-phosphoric acid. The volume of the sample injected was 20 l~L.The detector was a Jasco model 870 tuneable adsorbance detector, and the eluenl was monitored at 273 nm. Data acquisition, processing, and reporting were handled using a Spectra PhysicData Jet Integrator (Darmstadt-Kranicstein, Germany).

IS

Experimental EMB and ambucetamide (AMB)(Figure 2) were kindly provided by Hoechst Roussel Vet (Unterschleigheim, Germany) and by Professor W. Lambert, University of Gent (Gent, Belgium), respectively.Phenacetin (Figure 2) was purchased from Sigma (St. Louis, MO).All the solvents were of HPLCgrade and were obtained from commercial sources; water used was Milli Q| filtered.

a~

,..i

,-4

Figure 3. HPLC chromatogram of drug-free whole blood (A). HPLC chromatogram of a blood specimen (B).

Apparatus HPLC instrumentation. The HPLCsystem used a Jasco model 880-PU pump to deliver mobile phase at the flow-rate of 0.9 mL/min to a Waters Spherisorb ODS2 C18 5 t~m (150 mmx 4.6-ram i.d.) analyticalcolumn (Milford,]VIA).A Waters C18 Novapak Guard Pack precolumn was used to protect the analytical column. The mobile phase consisted of CH3OH/H20 (65:35,

1oo. 2 ~ 7

IZ7~

1348B 1 , t ~

Time ( m i n i

HBCO~

~/'~

CH3

~-(Dibutylamino)-4-methoxybenzeneacetamide

Ambucetamide H~C---x

'o-4/

F---x

\x,_

H

~-CHB 0

,o

N-(4-Ethoxyphenyl) acetamide

Phenacetin Figure 2. Molecular formula of the analytical marker ambucetamide and of the internal standard phenacetin.

324

.... "

~ "

~o~

"

~

=~, , ~ i o " t ~

"

I~ ~ ' " , : ~ o

"

~:~";s~o" ~:~; '

Time (mini

Figure 4. GC-MS chr0matogram of drug-free whole blood (A) and a blood specimen (B).

Journal of Analytical Toxicology, Vol. 25, July/August2001

GC-MS instrumentation. A Fison 8000 GC and a Fison MD 800 MS (Milan, Italy) were used for the quantitative analysis of EMB. This compound was separated on a Mega| OV-1 fusedsilica capillary column (15 m x 0.18-mm i.d., 0.1-mm film thickness; Milan, Italy). The injector was operated in the splitless mode at 250~ helium was used as carrier gas at a column head pressure of approximately 6 psi and a flow rate of 1 mL/min. Specimens (~1 mL) were injected at 100~ the split valve was opened after 1 min, and the temperature was increased to 295~ at a rate of 15~ When the temperature reached 295~ the course was extended for 10 rain. The ion source was operated at 180~ with an accelerating voltage of 70 eV, 750 mA filament current, and 1 kV electron multiplier voltage (the conversion @nodes operated at 5 kV). Ion current was acquired at the following mass-to-charge ratios: 121, 135, 190 (EMB) and 121, 192, 248 (AMB).

Methods

Samplepreparation. Quantitation of EMB concentration was based on a six-point calibration curve. Standard curves for EMB were prepared by adding EMB to pools of drug-free rat blood, liver, and kidney at concentrations of 2.5, 5, 10, 50, 250, and 500 pg/mL These samples were then taken through the analytical procedure. For the HPLC analysis, the peak areas of EMB relative to the internal standard (IS) were calculated and plotted versus the concentrations. An external standard procedure was followed for the extraction of the samples to assay by GC-MS analysis, and AMBwas used as an analytical marker for the determination of the exact volume of the specimen injected in the apparatus. The concentration of EMB in unknown samples was calculated from the calibration curves. Calibration graphs for blood, liver, and kidney were constructed also for EMB determination in SIM mode (data not shown). In this equation, x value was the concentration of EMB added to biological matrices in micrograms per milliliter, and y value was the peak area of the base peak (m/z 121). ~voohundred milligrams of NaHCO3 (in order to reach a pH between 8.5 and 9.0) and 8 mL of dichloromethane (CH2C12) containing 100 pg of phenacetin as IS (recovery 78 ___6%) were added to a 1.0 mL volume of blood. Phenacetin was added only for the HPLC sample preparations, whereas in extractions for GC-MS analysis an external standard procedure was performed (EMB recovery 92 _+4%). The extraction tubes were then placed on an oscillatory mixer (10 rain) at the speed of 60 oscillations/min. After centrifugation for 10 rain at 2000 x g at room temperature, the lower organic layer was transferred into a conical tube. The solvent was evaporated to dryness under a gentle stream of nitrogen without heating. The residue was redissolved in CHaOH (1 mL), vortex mixed, and transferred into 1.5 mL screw-capped vial. A 20-tJL aliquot was injected onto the HPLC; after the addition of the same volume of AMB solution (50 pg/mL), about 1 pL was injected onto the GC-MS system. A pool of tissue samples was homogenized with an Ultra

Table I. Accuracy and Precision Data for EMB Analysis in Whole Blood Method

Level

HPLC

Within.day 50 mg/mL 500 mg/mL

n

CV (%)

5 5

4.7 4.6

5 5

7.1 6.2

5 5

4.3 4.0

5 5

6.4 6.1

Day-to-day 50 mg/mL 500 mg/mL

Within-day

GC-MS

50 mg/mL 500 mg/mL

Day-to-day 50 mg/mL 500 mg/mL

Scan B+

AES-500 578 (11.637) Rf(1,10.000) 100]

121 I

98

t

89

J43

91 I

8zl

,

r

135

I

I

11TI

I 8.611 I'., 1o8"1I1.=

46

85

85

i

191 /

161 177 h82 I 147 1491[ I

92

3.2508

190

192

22

(.....~ ..... ,~ ~ .... ,...~...,.-.~1~..., ....100'.... ' ....120'.... ' ....1481 .... , ....180'.... ' ....180'....

' "'1200'" ,l"

220"1"" '

~6

1 " ' 2 4 0 " 1. . . .

292 ~4.276 i . . . . 2 5 0 1. . . .

i . . . . 2 8 0 1. . . .

293

1_295.3o8

i " " 3" 01 0" '

'1

mlz ,NVIBUA1 550(11.173) Rf(3,10.000)

Scan B+ 248

100] 1 44

121

"1 1 o:;

136

8.1506

._

9194 lO,9 i1~11., e

,?

11,1 ,,I 8Se6l,, 7e I./ lO41 I ~111,3 11.49 111.6s 19ol 204 s217 2s3246 25O oJill,... ~,11,.. ,g: ..... lit..,,,,. ~l,lL.,...,lk,I,.....,,lu,.,.,ll It...... ~E ......, I1( ............. h h....... I~'. ,/.................... 5 I''

40

'' I" "'

I''''1

60

"'''

I'''"

80

I ....

I''"

100

'1''''

I''''

120

I''''

I''''1''

140

''1''''

100

I''''

I" ' ' ' 1

180

....

I''''

200

I''''

I''''1''''1''''

220

240

I ', , , ,

i, ,, ,i,,

250

,,i,,,,

i,,

250

m/z Figure 5. Mass spectraof EMB and AMB.

325


Turrax mixer after dilution with water (1:1, w/w), and 2-g aliquots were extracted using the same protocol described for blood samples. The limit of detection (LOD) was defined as the smallest amount of the analyte that could be detected with a signal-tonoise ratio of 10:1. The limit of quantitation (LOQ) was estimated as five times the LOD. Reproducibility. Within-day reproducibility was evaluated by analyzing five blood samples spiked with EMB to obtain two different levels (50 and 500 pg/mL). Day-to-day reproducibility was determined by analysis of five replicate samples of the two levels (50 and 500 pg/mL) on seven consecutive days. From the data obtained, within-day and day-to-day correlation values (CV) were calculated. Drug administration. All the animals were administered by intravenous, intracardiac, and intrapulmonary route with the minimum dose recommended by the producer. From the dead animals, blood, liver, and kidney were excised and stored at -20~ until assay. All the organs were sampled in different parts before the analysis. Statistical analysis. Slope, intercepts, and correlation coeffiTable II. Ion Ratios for EMB and AMB Added to DrugFree Whole Blood at a Level of 5 pg/mL and Analyzed with GC-MS in the SIM Mode Average ion ratio (%)*

m/z190/121

m/z135/121

73.4 (1.6) 72.2 (1.6)

63.1 (1.3) 61.9 (1.3)

EMB Standard Fortified sample

m/z121/248

m/z192~248

AMB Standard Fortified sample

75.0 (2.3) 73.6 (2.3)

47.2 (0.9) 45.5 (1.0)

cient were determined for the calibration curve by linear regression (StatView). Comparison among mean values (blood, liver, and kidney) was performed by one-way analysis of variance for independent sample. When a significant overall difference was detected, a Dunnett test was used. A value ofp _