Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 60) ppt

For analysis of GLUF and GLYP in biomedical specimens, various methods by a modifi ed technique of the standard GC-NPD with N-acetyl and O-methyl derivatizations [4], GC/MS using tert-bu

© Springer-Verlag Berlin Heidelberg 2005

II.7.3 Glufosinate and

glyphosate

Introduction

Non-selective phosphorus-containing amino acid-type herbicides (PAAHs) to be used for foli-age exhibit lower toxicities than paraquat and are easily obtainable; they, thus, have come into wide use since 1980 Th e PAAHs include glufosinate (GLUF), glyphosate (GLYP) and biala-phos (BIAL) In Japan, there are many kinds of products containing GLUF and GLYP com-mercially available, and the number of suicidal cases using them is increasing [1]

In acute poisoning by GLUF, there is a latent period for 4–60 h before appearance of poi-soning symptoms, such as lowered consciousness levels, respiratory arrest and generalized convulsion; when more than 100 mL of BASTA Fluid® (GLUF, 18.5 %; anion surfactant; blue-green in color) is ingested, the physical conditions of the victim are seriously aggravated with high incidence [2] Respiratory controls, such as securance of the respiratory tract and artifi cial respiration, are very important for rescuing such victims Since it is possible to predict the aggravation of the GLUF poisoning for a victim from the time aft er its ingestion and from a blood GLUF concentration [3], the rapid analysis of blood GLUF becomes very meaningful not to miss the timing for starting the respiratory control; it is critical to prevent a victim from falling into the unfortunate turning point

For analysis of GLUF and GLYP in biomedical specimens, various methods by a modifi ed

technique of the standard GC-NPD with N-acetyl and O-methyl derivatizations [4], GC/MS using tert-butyldimethylsilyl (t-BDMS) derivatization [5–7], TLC [8], HPLC with fl uorescence detection aft er post-column derivatization using o-phthalaldehyde [9], HPLC with fl

uores-cence detection aft er pre-column derivatization using 9-fl uorenylmethyl chloroformate (FMOC-Cl) [10], HPLC with UV detection aft er pre-column derivatization using phenyl iso-thiocyanate [11], ion chromatography with electrochemical detection without any

derivatiza-tion [12], LC/MS with N-acetyl and O-methyl derivatizaderivatiza-tions [13] and HPLC with UV detec-tion aft er pre-column derivatizadetec-tion using p-nitrobenzoyl chloride [14] were reported.

In this chapter, some details on GC/MS [7], HPLC with fl uorescence detection [10] and HPLC-UV [14], aft er each derivatization for analysis of PAAHs, are described

GC/MS analysis [7]

Reagents and their preparation

• GLUF (DL-homoalanin-4-yl(methyl)phosphinate monoammonium salt) and its

metaboli-te 3-methylphosphinicopropionic acid (MPPA) can be purchased from Wako Pure

Chemi-cal Industries, Ltd., Osaka, Japan; GLYP (N-(phosphonomethyl)glycine) and its metabolite

aminomethyl phosphonic acid (AMPA) from Sigma (St Louis, MO, USA) Th eir chemical structures are shown in > Figure 3.1 Each compound was dissolved in 10 % methanol

aqueous solution; they are stable at least for 6 months under refrigeration Th e trace amounts of these compounds can adsorb to glassware; when low levels of the compounds are dealt with, the tools made of Tefl on should be used [13]

• DL-2-Amino-3-phosphonopropionic acid (APPA) purchased from Aldrich (Milwaukee,

WI, USA) is used as internal standard (IS)a and dissolved in 10 % methanol aqueous solu-tion to prepare its 100 µg/mL solution

• N-Methyl-N-(tert-butyldimethylsilyl)trifl uoroacetamide (MTBSTFA) and

N,N-dimethyl-formamide can be purchased from Aldrich and should be stored in a dry state (not to be contaminated by water)

• 0.1 M NaOH solution: the 1 M solution of reagent grade is diluted 10-fold with distilled water

• To construct calibration curves, various amounts of GLUF, MPPA, GLYP or AMPA to-gether with a fi xed amount of APPA (IS) are spiked into the extracts of the standard human serum, evaporated to dryness and derivatized before analysisb

GC/MS conditions

Instrument: a GC 17A gas chromatograph/a QP5050 mass spectrometer (Shimadzu Corp., Kyoto, Japan); column: DB-5MS (15 m × 0.25 mm i.d., fi lm thickness 0.25 µm, J&W Scientifi c, Folsom, CA, USA); column temperature: 80 °C (2 min) → 15 °C/min → 300 °C (5 min); carrier gas: He; its fl ow rate; 1.0 mL/min; injection: split/splitless mode (splitless for 2 min); split ratio: 10; injection amount: 1 µL; injection temperature: 300 °C; interface temperature: 280 °C;

ion-ization mode: EI; scan range: m/z 70–650.

Structures of glufosinate (GLUF) and glyphosate (GLYP) and their metabolites.

⊡ Figure 3.1

Procedure

i As a specimen, serum, urine or stomach contents are used; 500 µL of undiluted serum or

500 µL of urine diluted 10-fold with distilled water is subjected to the following procedure

ii Th e above specimen is mixed with 500 µL acetone, vortex-mixed and centrifuged (3,000 rpm,

5 min) for deproteinizationc; 100 µL of the supernatant solution is subjected to the next step For stomach contents, they are diluted with distilled water appropriately and pass through a membrane fi lter (0.45 µm); 100 µL of the fi ltrate is subjected to the next step

iii Isolute® HAX 100 mg cartridges (International Solvent Technology, Mid Glamorgan, UK)d, which have anion exchanging and hydrophobic interaction properties, are used for extraction One of the cartridges is activated by passing 1 mL methanol, 1 mL of 0.1 M NaOH solution and 1 mL distilled water through it at a fl ow rate of 1 mL/min

iv Th e above 100 µL specimen solution is mixed well with 10 µL of IS solution and 1 mL dis-tilled water, and poured into the cartridge Th e pH of the solution should be 6.4–8.5; there-fore, it is generally not necessary to adjust pH for the serum or urine specimens

v Th e cartridge is washed with 1 mL distilled water, and a target compound and IS are eluted with 500 µL of 1 M HCl solution/methanol (4:1) at a fl ow rate of 500 µL/min Th e eluate is evaporated to dryness under reduced pressure with warming at 50 °C

vi Th e residue is mixed with 50 µL MTBSTFA and 50 µL N,N-dimethyl-formamide, son icated

for 2 min and heated at 80 °C for 30 mine for t-BDMS derivatization.

vii Aft er cooling to room temperature, 1 µL of the fi nal solution is injected into GC/MS

Assessment of the method

In this method, solid-phase extraction was used to extract a PAAH and its metabolite in a

bio-medical specimen for their GC/MS analysis [15] aft er t-BDMS derivatization > Figure 3.2

shows mass spectra of t-BDMS derivatives of GLUF, MPPA, GLYP, AMPA and APPA Th e base

peaks at m/z M–57 appear for all compounds Th e quantitation using the selected ion

monitor-ing (SIM) is made with each base peak (GLUF: m/z 466; MPPA: m/z 323; GLYP: m/z 454; AMPA: m/z 396; APPA: m/z 568).

Various derivatization methods were reported for PAAHs [16]; the advantage of the use of

t-BDMS derivatization is the one-step reactionf , which completes in only 30 min When pg levels of PAAHs are derivatized with high effi ciency, the N-acetyl and O-methyl derivatizations

using acetic acid and trimethyl orthoacetate are useful [17]

Th e detection limit for both GLUF and GLYP in the scan mode is about 100 pg on-column (about 0.1 µg/mL in bood); that of both MPPA and AMPA is about 10 pg on-column In the SIM mode, the CV values refl ecting reproducibility for the 4 compounds (100 ng each for

de-rivatization) using APPA as IS are not larger than 3 % (n = 5); GLUF and GLYP show linearity

in the range of 100 pg–100 ng on-column Th e detection limit (S/N ratio = 5) of GLUF and GLYP in the SIM mode is about 10 pg on-column; that of MPPA and AMPA is even lower

Th e recovery rates for GLUF and GLYP, which had been spiked into sera at a concentration

of 1 µg/mL, aft er extraction with the Isolute® HAX cartridge, were as good as 93.3 ± 6.7 %

(n = 5) and 92.6 ± 7.2 % (n = 5), respectively Upon extraction with the cartridge, a urine

spec-imen should be diluted suffi ciently, because in the presence of strong anions in a specimen, the recovery rate becomes low Since unchanged forms of GLUF and GLYP are rapidly excreted

GC/MS analysis

EI mass spectra of t-BDMS derivatives of GLUF, MPPA, GLYP, AMPA and APPA (IS).

⊡ Figure 3.2

TIC (upper panel) and SIM chromatograms (lower panel) obtained by GC/MS for t-BDMS

derivatives of GLUF, MPPA, GLYP, AMPA and APPA (IS) (10 ng each on-column).

⊡ Figure 3.3

TIC (upper panel) and SIM chromatograms (lower panel) obtained by GC/MS for an extract of

serum of a patient, who had ingested a GLUF-containing herbicide.

⊡ Figure 3.4

GC/MS analysis

into urine, there are many cases, in which they are suffi ciently detectable even from 100-fold diluted urine

> Figure 3.3 shows a TIC and SIM chromatograms for t-BDMS derivatives of the

authen-tic GLUF, MPPA, GLYP, AMPA and APPA (IS); > Figure 3.4 shows comparable

chromato-grams for the extract of serum, which had been obtained from a poisoned victim 7 h aft er in-gestion of 80 mL of a GLUF product (BASTA Fluid®) Using the base peaks at m/z M–57,

GLUF and MPPA could be specifi cally detected by SIM from the extract of the crude matrix obtained from the actual case; the concentrations of GLUF and MPPA were 74.3 and 0.32 µg/mL, respectively

HPLC analysis with fluorescence detection [10]

Reagents and their preparation

• FMOC-CL is purchased from Sigma, and dissolved in acetone to prepare its 0.1 % solution just before use

• Borate buff er solution (0.1 M, pH 8.5): 2 g of sodium tetraborate is dissolved in 100 mL distilled water and the pH is adjusted to 8.5 with 2 M HCl solution

• Phosphate buff er solution (10 mM, pH 2.5): 240 mg of sodium dihydrogenphosphate is dissolved in 200 mL distilled water and the pH is adjusted to 2.5 with phosphoric acid

HPLC conditions

Instruments: an LC-10ADVP pump, a CTO-10ACVP column oven, an RF10AXL fl uoro-photometer, an SIL-10ADVP autosampler, a CLASS-VP analysis soft ware (all from Shimadzu Corp.); column: Inertsil® ODS-2 (150 × 4.6 mm i.d., particle size 5 µm, GL Sciences, Tokyo, Japan); column temperature: 40 °C; mobile phase: acetonitrile/10 mM phosphate buff er solu-tion (ph 2.5); gradient elusolu-tion: the ratio of the above acetonitrile and 10 mM phosphate buff er solution of the mobile phase is held at 3:7 (v/v) for 7 min, and changed to 1:1 aft er 13 min and

to 8:2 aft er 15 min (1 min-hold) (the gradient elution aft er 13 min is conducted for washing the column); fl ow rate of the mobile phase: 1 mL/min; detector: a fl uorophotometer; excitation wavelength: 265 nm; emission wavelength: 315 nm

Procedure

i A 100-µL volume of undiluted serum or urine diluted 10-fold with 0.1 M borate buff er solution (pH 8.5) is mixed with 400 µL of 0.1 M borate buff er solution and 1 mL acetone

ii Th e above solution is vortex-mixed and centrifuged at 3,000 rpm for 5 min for depro-teinization; the resulting supernatant solution is subjected to the below derivatization iii A 50-µL volume of the above solution is mixed with 200 µL of 0.1 M borate buff er solution (pH 8.5) and 200 µL of 0.1 % FMOC-Cl acetone solution, capped and mixed well g Th e mixture is incubated at 40 °C for 10 min

iv A 500-µL volume of ethyl acetate is added to the mixture and shaken to remove excessive FMOC-Cl A 100-µL aliquot of the aqueous layer is mixed with 400 µL of 0.1 M borate buff er solution (pH 8.5); 10 µL of it is injected into HPLC

Assessment of the method

> Figure 3.5 shows HPLC chromatograms for the authentic standard solutions of GLUF and

GLYP (10 µg/mL) and for an extract of serum sampled 8 h aft er ingestion from an actual poi-soned patient, who had ingested 25 mL of BASTA Fluid®

GLUF and GLYP are highly polar in their unchanged forms, and thus suitable for separa-tion by ion-exchange chromatography Aft er derivatizasepara-tion with FMOC-Cl, the compounds become separable by reversed phase HPLC using an acetonitrile-phosphate mobile phase

Th e derivatization can be completed under mild conditions at 40 °C for 10 min; the deriva-tives are stable for at least 19 h Th e detection limit is as low as about 1 ng/mL in a specimen; the whole procedure is accomplished in about 40 min

⊡ Figure 3.5

Chromatograms obtained by HPLC with fluorescence detection for the authentic GLUF and GLYP (upper panel) and for an extract of serum of a patient, who had ingested a GLUF-containing

herbicide (lower panel), after derivatization with FMOC-Cl.

HPLC analysis with fl uorescence detection

Th e method with fl uorescence detection is higher in both specifi city and sensitivity than that with UV detection Th ere is another report [9] dealing with HPLC with fl uorescence

de-tection of PAAHs, in which post-column derivatization with o-phthalaldehyde is employed

However, it requires a special device for post-column derivatization By the method described

in this section, PAAHs can be simply measured only by combining usual reversed phase HPLC with a fl uorescence detector

HPLC analysis with UV absorption detection

Reagents and their preparation

• p-Nitrobenzoyl chloride (PNBC) can be obtained from Aldrich (Milwaukee, WI, USA)

and other manufacturers; it is dissolved in acetonitrile (of the highest purity)h to prepare

1 % solution just before use

• Borate buff er solution (0.1 M, pH 8.5): 2 g of sodium tetraborate is dissolved in 100 mL distilled water and the pH is adjusted to 8.5 with 2 M HCl solution

• Ammonium acetate solution (10 mM, pH 5): 154 mg of ammonium acetate (of the highest purity) is dissolved in 200 mL of ultra-pure distilled water and its pH is adjusted to 5 with acetic acid

HPLC conditions

Instruments: the same pump, column oven, autosampler and soft ware as described in the sec-tion of HPLC analysis with fl uorescence detecsec-tion, and an SPD-M10AVP diode array detector (all from Shimadzu Corp.); column: Inertsil® Ph-3 (150 × 4.6 mm i d., particle size 5 µm, GL Sciences); column temperature: 40 °C; mobile phase: acetonitrile/10 mM ammonium acetate solution (pH 5.0) (1:9, v/v); fl ow rate: 0.8 mL/min; detection wavelength: 272 nm

Procedure

i A 500-µL volume of undiluted serum or urine diluted 10-fold with distilled water is mixed with 500 µL acetone, vortex-mixed and centrifuged at 3,000 rpm for 5 min for deprotein-ization

ii A 100-µL aliquot of the above supernatant solution is mixed with 200 µL of 0.1 M borate buff er solution (pH 8.5) and 100 µL of 1 % PNBC acetonitrile solution, and left at 22–25 °C for 10 min; 10 µL of the solution is injected into HPLC

Assessment of the method

Th e advantages of this method are that the derivatization reaction is completed in 10 min at room temperature and that a usual reversed phase HPLC-UV detection can be used; with the

minimum instruments and time, the screening and quantitation of GLUF and GLYP can be achieved

> Figure 3.6 shows HPLC chromatograms for the authentic GLUF and GLYP and for the

extract of serum, into which GLUF and GLYP had been spiked Since the polarity of the com-pounds is high even aft er derivatization, their peaks appear at early retention times by the re-versed phase HPLC Th e peak 3 shown in the upper chromatogram of > Figure 3.6

corre-sponds to the unreacted reagent (PNBC); the peak 4 to p-nitrobenzoic acid formed from PNBC

by its reaction with water Th e λmax wavelengths for the derivatives of GLUF and GLYP are

272.8 and 273.1 nm, respectively (> Figure 3.7).

When a usual ODS column is used, the GLYP peak appears without any interference, but the GLUF peak may be interfered with by impurity peaks derived from the crude matrix By using the Inertsil® Ph-3 column, which includes phenyl groups for their interaction with the target compounds, the GLUF peak can be better separated from impurities

Th e detection limit for both authentic GLUF and GLYP in clean solution is 0.01 µg/mL; while that for GLUF and GLYP in serum or urine is 0.1 µg/mL Th e average recovery of GLUF from sera at the concentration of 1.0 µg/mL was as good as 95.3 % (n = 5); that from urine at

10.0 µg/mL 97.3 % (n = 5).

Chromatograms obtained by HPLC with UV absorption detection for the authentic GLUF and

GLYP (a) and for an extract of serum (b), into which GLUF and GLYP had been spiked, after

derivatization with PNBC Peaks 3 and 4 are due to the unreacted reagent (PNBC) and a

by-product (p-nitrobenzoic acid), respectively.

⊡ Figure 3.6

HPLC analysis with UV absorption detection

Toxic and fatal concentrations

GLUF [18]

Th e number of poisoning cases by GLUF counts 100–200 per year; most cases are due to suicidal ingestion of GLUF products Its main products are BASTA Fluid® (GLUF, 18.5 % anion surfactant,

30 %; blue-green color) and Hayabusa® (GLUF, 8.5 %; anion surfactant, 50 %; blue color)

Th e oral LD50 values (mg/kg) for GLUF are 1,660/1,510 (male/female) in rats and 436/464

in mice In humans, the poisoning symptoms become severe, when more than 100 mL of the 18.5 % solution of GLUF is ingested Th e oral LD50 value (mg/kg) for the anion surfactant being contained in the BASTA Fluid® is 4,500 in rats

In GLUF poisoning, there is a characteristic latent period without any symptom lasting for not less than 6 h; aft er this period, poisoning symptoms, such as lowering of the consciousness level, respiratory suppression and generalized convulsion, suddenly appear Th e severity of the GLUF poisoning can be predicted by plotting the time aft er ingestion on the horizontal axis and the logarithm of serum GLUF concentration on the vertical axis Koyama et al [2] mea-sured serum GLUF levels in 99 patients with GLUF poisoning, and drew two linear lines A and

B by connecting a point of 70 µg/mL at 2 h aft er GLUF ingestion with a point of 5 µg/mL at 8 h for A and by connecting a point of 200 µg/mL at 2 h with that of 15 µg/mL at 8 h for B Th ey reported that any plot below line A indicated a mild case, and one above line B a severe case; in the area between lines A and B mild and severe cases were mixed Both GLUF and coexisting surfactant seem exerting toxic eff ects in many GLUF poisoning cases

GLUF shows contradictory eff ects on the central nervous system, viz., its excitation

and suppression; it may act on glutamate synthase, glutamate decarboxylase and inhibitory glutamic acid receptors Th e surfactant contained in the GLUF product is being considered responsible for vomiting, erosion of the upper digestive tracts, edema appearing from the oral mucosa to the larynx and shock accompanied by the peripheral resistance

UV absorption spectra for GLUF and GLYP after derivatization with PNBC.

⊡ Figure 3.7