Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 51) doc

Assessment of the method Aconite toxins can exert their toxic eff ects at very low concentrations, which means that their concentrations in human body fl uids are very low.. Poisoning ca

© Springer-Verlag Berlin Heidelberg 2005

by Michinao Mizugaki and Kitae Ito

Introduction

Th e aconite plants contain Aconitum alkaloids (AAs) and other minor components, such as

chasmanine, kobusine and higenamine AAs consist of aconitines, benzoylaconines and aco-nines as shown in > Figure 1.1 Th e most toxic group is the aconitines, including aconitine, mesaconitine, hypaconitine, and jesaconitine; this group is one of the most poisonous com-pounds being contained in the plant kingdom

Even nowadays, aconite poisoning cases take place occasionally Th ese may be due to ac-cidental, suicidal or homicidal ingestion of the plant itself or its extracts

In this chapter, a specifi c method for GC/MS analysis of AAs in human specimens is pre-sented

Reagents and their preparation

• Aconitine was purchased from Sigma (St Louis, MO, USA); mesaconitine and hypaconi-tine from Kishida Kagaku (Osaka, Japan) Jesaconihypaconi-tine, benzoylaconine, benzoylmesaco-nine, benzoylhypacobenzoylmesaco-nine, 14-anisoylacobenzoylmesaco-nine, acobenzoylmesaco-nine, mesaconine and hypaconine were donated by Tsumura (Tokyo, Japan) Benzoylaconine and aconine can be obtained also

Structures of Aconitum alkaloids (AAs).

⊡ Figure 1.1

from Sanwa Shoyaku (Tochigi, Japan) As a trimethylsilylating reagent, N,O-bis(trime

thyl-silyl)trifl uor o acet amide (BSTFA), containing 1 % trimethylsilylchlorosilane (TMCS)a (Pierce, Rockford, IL, USA) is used Pyridine to be used is of amino acid sequence analysis grade (Wako Pure Chemical Industries, Ltd., Osaka, Japan or Sigma) Other organic solvents are

of HPLC grade; other reagents are of special grade

• Each compound of aconitines, benzoylaconines and aconines is dissolved in acetonitrile to prepare the 1 µg/mL standard solution separately

Instrumental conditions

i Instrument

A DX-303 GC/MS instrument (JEOL, Tokyo, Japan) equipped with a Van den Berg type solvent-less injection device and a DA-5000 data processor (JEOL)

ii GC/MS conditions

Ionization: EI; ion source temperature: 250 °C; electron energy: 70 eV; trap current: 0.3 mA; accelerating voltage: 3 kV; GC column: a DB-5 chemical bond fused silica capillary column (15 m × 0.25 mm i d., fi lm thickness 0.25 µm, J&W Scientifi c, Folsom, CA, USA); column (oven) temperature: 250 °C→16 °C/min→320 °C; injection temperature: 320 °C; carrier gas:

He; its fl ow rate: 25 m/min (linear velocity at 250 °C); scan range: m/z 100–800; scan speed:

1.2 s (repetition time, 2 s); resolution: 1,000

iii Selected ion monitoring (SIM)

Th e instrumental conditions are the same as above Each base peak of the trimethylsilyl (TMS) derivatives of the alkaloids is used for SIM For hypaconitine, mesaconine, aconitine and

jesa-conitine, peaks at m/z 596, 684, 698 and 728 are used, respectively For benzoylhypaconine, benzoylmesaconine and benzoylaconine, those at m/z 686, 774 and 788 are used, respectively; for hypaconine, mesaconine and aconine, those at m/z 654, 742 and 756 are used as monitoring

ions, respectively

Procedures

i Construction of the calibration curve

i For plotting diff erent concentrations, 5, 10, 25, 50 and 100 µL aliquots of each AA standard solution (1 µg/mL) are placed in glass vials

ii Th ey are evaporated to dryness under a stream of nitrogen

iii Each residue is dissolved in 50 µL pyridine, followed by addition of 50 µL of 1 % TMCS-BSTFA and being capped airtightly

iv Th e derivatization reaction is made by leaving the mixture vials overnight at room temper-ature b

v A 1-µL aliquot of each solution is injected into GC/MS

vi Peak areas are plotted against compound concentrations to construct an external calibra-tion curve

457 Aconite toxins

ii Extraction and derivatization procedure

i A 1.0-mL volume of a body fl uid specimen (whole blood, serum or urinec) and 10 mL methanol d are placed in a glass test tube and mixed well for deproteinization

ii It is centrifuged at 3,000 g for 10 min to obtain supernatant solution.

iii To the sediment, 10 mL methanol is again added, mixed well and centrifuged to obtain the second supernatant solution

iv Th e supernatant solutions are combined and evaporated to dryness at 40 °C under re-duced pressure

v Th e residue is dissolved in 0.25 mL acetonotrile and mixed well

vi A Bond Elut SI cartridge (Analytical International, Harbor City, CA, USA) is equilibrated with 5 mL n-hexane

vii Th e above acetonitrile solution is poured into the silica gel cartridge

viii To the container (or a test tube) at the step v), 0.25 mL acetonitrile is again added to rinse

it well; the solution is also poured into the same cartridge

ix Th e cartridge is washed with 10 mL chloroform

x It is further washed with 10 mL ethyl acetate

xi A 20-mL volume of diethylamine/chloroform (1:1) is passed through the cartridge to elute AA compounds

xii Th e eluate is evaporated to dryness under reduced pressure

xiii Th e residue is dissolved in 50 µL pyridine, followed by the addition of 50 µL of 1 % TMCS-BSTFA for derivatization at room temperature overnight e A 1-µL aliquot of the resulting solution is injected into GC/MS

xiv Th e peak area of each AA obtained by SIM for a specimen is applied to the above calibra-tion curve to calculate its concentracalibra-tion

Assessment of the method

Aconite toxins can exert their toxic eff ects at very low concentrations, which means that their concentrations in human body fl uids are very low Th erefore, it was diffi cult to identify and quantitate them by the conventional HPLC method, because of its low sensitivity and spe-cifi city

In this chapter, a sensitive GC/MS method for simultaneous analysis of AAs, including aconitines [1], benzoylaconines and aconines, has been presented [2] Th is method allows accurate and simultaneous quantitation of AAs in small volumes of specimens

i EI mass spectra of AA-TMS derivatives

and aconines

• TMS derivatives of aconitines

By the derivatization with the above reagents, aconitine, mesaconitine and jesaconitine give bis-TMS derivatives; while hypaconitine a mono-TMS derivative In each mass spec-trum, a [M–CH3COOH–OCH3]+ ion appears as the base peak; a few small fragment peaks

also appeared (> Figure 1.2).

• TMS derivatives of benzoylaconines

Benzoylaconine and benzoylmesaconine give tri-TMS derivatives; while benzoylhypaco-nine a bis-TMS derivative In each mass spectrum, a [M–OCH3]+ ion appears as the base

peak; other fragment peaks are a few and small (> Figure 1.3).

• TMS derivatives of aconines

Aconine and mesaconine give tetra-TMS derivatives; while hypaconine a tri-TMS deriva-tive In each mass spectrum, a [M–OCH3]+ ion appears as the base peak (> Figure 1.4).

As shown in > Figures 1.2–1.4, the ratio of each base peak to the total abundance is

rela-tively high; the base peaks are very useful for trace quantitative analysis by GC/MS-SIM

ii Reliability of the method

of 9 kinds of aconitines and their hydrolysis products had been spiked For every compound,

a sharp peak f appeared at the same retention time as that of the authentic one; no interfering impurity peaks were observed

Th e calibration curve consisting of peak area on the vertical axis and compound amount

(in an injected volume) on the horizontal axis showed good linearity (r2 = 0.999) in the range

of 100 pg–7.5 ng for each alkaloid Th e detection limits by this method were about 10 pg (S/N 10)

in an injected volume, enabling highly sensitive analysis

Th e recoveries of 50 ng each of AAs, which had been spiked into 1 mL human serum, were 85.2–94.4 % Th e above pretreatment procedure can be applied to urine, whole blood and tissue g specimens In addition, the authors synthesized d5-aconitine for use as internal stan-dard (IS), resulting in higher precision and sensitivity in aconitine analysish [3]

Poisoning cases, and toxic and fatal concentrations

Th e aconitines are neurotoxic and cardiotoxic; they exert toxic eff ect by acting on the gate mechanisms of sodium-ion channels of cell membranes and by causing hyperpolarization

of cells [4] One of the most important poisoning symptoms is arrythmia Arrythmia is sometimes changed to ventricular fi brillation, and cardiac and respiratory arrest resulting in death [5]

Th e history of aconite as a poison is very long, and dates back to ancient times [6, 7] However, no reports on the concentrations of the aconite toxins (aconitine, mesaconitine, hypaconitine and jesaconitine) were available, because of the low sensitivity of analytical methods

Now, the authors are undertaking the analysis of AAs in body fl uids of aconite-poisoned patients in every area of Japan, and thus accumulating the data [8–14] > Table 1.1 shows the

concentrations of AAs in blood and urine in 6 poisoning cases

A 50-year-old female attempted suicide by crunching and ingesting one and a half of thumb-sized tubers of aconite, and visited a doctor 30 min later She had had a history of de-pression and had received psychiatric treatments Th e numbness of the tongue appeared in the midst of her eating; but her consciousness was clear and notifi ed the doctor of her ingestion of aconite for the purpose of suicide Her blood pressure was 100/60 mmHg; her heart beat 110/ min and arrythmic She complained of nausea, vomiting and the numbness of her whole body;

EI mass spectra for TMS derivatives of aconitines (a): aconitine; (b): mesaconitine;

(c) hypaconitine; (d): jesaconitine.

⊡ Figure 1.2

Poisoning cases, and toxic and fatal concentrations

EI mass spectra for TMS derivatives of benzoylaconines (a): benzoylaconine; (b) benzoylmasaco-nine; (c): benzoylhypaconine.

⊡ Figure 1.3

EI mass spectra for TMS derivatives of aconines (a) aconine; (b): mesaconine; (c): hypaconine.

⊡ Figure 1.4

Poisoning cases, and toxic and fatal concentrations

SIM chromatograms for TMS derivatives of AAs Nine kinds of AAs (50 ng each) were spiked into

1 mL serum, and extracted according to the procedure; a 5-µL aliquot of the final extract solution (50 µL) was injected into GC/MS.

⊡ Figure 1.5

she could not stand up by herself Her electrocardiogram showed her continuing arrythmia; the basic rhythm of her heart beat was the sinus one at about 100/min Multiple-sourced and frequent ventricular arrythmia, atrial arrythmia, intraventricular aberrant conduction, QT elongation and torsades de pointes were observed

Transfusion, gastrolavage and administration of activated charcoal and a purgative were performed For the arrythmias, lidocaine and disopyramide were administered at an early stage, but were not eff ective Th us, the administration of phenytoin was started, but unsuccess-ful Th e blood pressure was lowered about 1 h aft er ingestion, and her respiration was arrested about 2.5 h aft er; artifi cial respiration, aft er endotracheal intubation, was started Th ereaft er, although administration of lidocaine, disopyramide and phenytoin was continued, they were not eff ective From about 7 h aft er ingestion, ventricular tachycardia continuing for about 10 s began to take place frequently Th iopental was administered, because of excitement and vigor-ous movement of her body Aft er the administrations of magnesium sulfate and propranolol, the arrythmias markedly decreased about 11 h aft er ingestion Th ereaft er, her conditions were improved smoothly She was extubated about 24 h aft er and discharged on day 6 without any sequela Th e concentration values of AAs in this case are shown as the case No 1 in

As shown in the above case, in the typical aconite poisoning case, the oral numbness just aft er ingestion extends to whole body, followed by hypotension and various types of arryth-mias; in the worst case, the arrythmias are aggravated into ventricular fi brillation and fi nally death

C No

Blood conc

S time af

ingestion (h)

Urine conc

(ng/ mL)

S time af

ingestion (h)

2.4 1.0

285 74.6

F F

Poisoning cases, and toxic and fatal concentrations

ii Lethal doses of aconitines

Th e LD50 values in mice for AAs are shown in > Table 1.2 According to the experiments for

aconitines using mice, the toxicity is highest for jesaconitine, followed by aconitine, mesaconi-tine and hypaconimesaconi-tine [15] Th e oral lethal doses of aconitine for humans were reported to be 1–2 mg [16, 17]

About 500 species of aconite genus plants are growing in the world Even in the same spe-cies, the composition ratio of the alkaloids and their contents diff er according to seasons and growing areas Th e contents of aconitines in the plant expressed per weight is highest in the tubers, followed by the fl owers, leaves and stems [15] Th e authors measured contents of

acon-itines in the tubers of Aconitum species harvested at a mountain in Nishi-Shirakawa-gun,

Fu-kushima Prefecture, according to 4 seasons; the total contents of aconitines per g were 2–4 mg

on average [18]

Th e cases Nos 2 were fatal; their concentrations of AAs in blood and urine were higher than those in the survived Nos 1 and 5 cases Like in the case No 6 of mistaken eating of aco-nite for an edible wild plant, the detection of AAs are sometimes diffi cult at relatively a long time aft er ingestion

When the blood concentrations were compared with those in urine, the latter generally gave higher AA values In the case No 5, his body fl uids could be sampled for analysis according to various time intervals aft er ingestion; the AAs could not be detected from blood as early as on the 2nd day, but some of the AAs could be detected from urine even on the 7th day [13] AAs in blood disappear in a relatively short period, but they are excreted into urine in rela-tively large amounts continuously Th erefore, even if a relatively long time elapses aft er inges-tion in a suspicious case of aconite poisoning, it is useful to analyze urine specimens

Th e hydrolytic compounds of aconitines (benzoylaconines and aconines) are being sug-gested to be metabolites of aconitines [13]

Th ere are various poisoning symptoms observable in aconite poisoning; the relationship between the appearance of the symptoms and AA concentrations in body fl uids remains to be explored

Th e distribution of AAs in human organs obtained at autopsy for the victim in the case

No 4, who had died 4 h aft er ingestion, is shown in > Table 1.3 Th e AA concentrations ex-pressed as ng/g wet weight were highest in the right lobe of the liver, followed by the left lobe of

⊡ Table 1.2

LD 50 values of AAs in mice (mg/kg)

The LD50 values were calculated by the up and down method The value for aconine was cited from reference [7]; for

the liver, kidney, heart, right lung, left lung, psoas major, adipose tissue (around psoas major), cerebellum and cerebrum Especially in right and left lobes of the liver and the kidney, very high concentrations of AAs were found Th e ratio of jesaconitine concentration in a organ to that in serum was not less than 3 for the right and left lobes of the liver and the kidney, showing the accumulation of AAs in the organs On the other hand, such ratio was low for psoas major, adi-pose tissue, cerebellum and cerebrum; it was only 0.03 for the latter two organs Th ese results suggest that the liver and kidney are useful for analysis of AAs in fatal poisoning cases

the case No 4 (> Table 1.1) [12] Th ey were highest in the ileum contents, followed by bile, jejunum contents, stomach contents and duodenum contents

Th e high AA levels found in the kidney is in accordance with the high levels in urine On the other hand, the high AA levels in bile and the contents of the ileum and jejunum show another

excretion route for AAs via the digestive tract into feces in addition to the urinary route.

⊡ Table 1.3

Distribution of AAs in human organs in Case 4

AAs other than the above compounds were below the detection limits The tissue/serum ratios are the data only for jesaconitine ND: below the detection limit.

⊡ Table 1.4

AA concentrations in the contents of digestive tracts in Case 4

jesa-conitine

14-anisoyl-aconine (ng/g)

benzoyl-aconine

ileum

contents

jejunum

contents

stomach

contents

duodenum

contents

Poisoning cases, and toxic and fatal concentrations