7. 3 7. 3 © Springer-Verlag Berlin Heidelberg 2005 II.7.3 Glufosinate and glyphosate by Yasushi Hori and Manami Fujisawa 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. 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 arti 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 a 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 modi 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 uorescence detection a er post-column derivatization using o-phthalaldehyde [9], HPLC with uores- cence detection a er pre-column derivatization using 9- uorenylmethyl chloroformate (FMOC-Cl) [10], HPLC with UV detection a 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 derivatizations [13] and HPLC with UV detec- tion a er pre-column derivatization using p-nitrobenzoyl chloride [14] were reported. In this chapter, some details on GC/MS [7], HPLC with uorescence detection [10] and HPLC-UV [14], a 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 546 Glufosinate and glyphosate aminomethyl phosphonic acid (AMPA) from Sigma (St. Louis, MO, USA). 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. e trace amounts of these compounds can adsorb to glassware; when low levels of the compounds are dealt with, the tools made of Te 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)tri 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 xed amount of APPA (IS) are spiked into the extracts of the standard human serum, evaporated to dryness and derivatized before analysis b . 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., lm thickness 0.25 µm, J&W Scienti c, Folsom, CA, USA); column temperature: 80 °C (2 min) → 15 °C/min → 300 °C (5 min); carrier gas: He; its 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 3.1 547 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. e above specimen is mixed with 500 µL acetone, vortex-mixed and centrifuged (3,000 rpm, 5 min) for deproteinization c ; 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 lter (0.45 µm); 100 µL of the 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 ow rate of 1 mL/min. iv. 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. 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. 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 ow rate of 500 µL/min. e eluate is evaporated to dryness under reduced pressure with warming at 50 °C. vi. 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 min e for t-BDMS derivatization. vii. A er cooling to room temperature, 1 µL of the 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] a er t-BDMS derivatization. > Figure 3.2 shows mass spectra of t-BDMS derivatives of GLUF, MPPA, GLYP, AMPA and APPA. e base peaks at m/z M–57 appear for all compounds. 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 reaction f , which completes in only 30 min. When pg levels of PAAHs are derivatized with high e ciency, the N-acetyl and O-methyl derivatizations using acetic acid and trimethyl orthoacetate are useful [17]. 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 re 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. 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. e recovery rates for GLUF and GLYP, which had been spiked into sera at a concentration of 1 µg/mL, a 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 su 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 548 Glufosinate and glyphosate EI mass spectra of t-BDMS derivatives of GLUF, MPPA, GLYP, AMPA and APPA (IS). ⊡ Figure 3.2 549 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 550 Glufosinate and glyphosate into urine, there are many cases, in which they are su 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 a 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 speci 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 bu 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 bu 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 uoro- photometer, an SIL-10ADVP autosampler, a CLASS-VP analysis so 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 bu er solu- tion (ph 2.5); gradient elution: the ratio of the above acetonitrile and 10 mM phosphate bu er solution of the mobile phase is held at 3:7 (v/v) for 7 min, and changed to 1:1 a er 13 min and to 8:2 a er 15 min (1 min-hold) (the gradient elution a er 13 min is conducted for washing the column); ow rate of the mobile phase: 1 mL/min; detector: a 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 bu er solution (pH 8.5) is mixed with 400 µL of 0.1 M borate bu er solution and 1 mL acetone. ii. 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 bu er solution (pH 8.5) and 200 µL of 0.1 % FMOC-Cl acetone solution, capped and mixed well g . e mixture is incubated at 40 °C for 10 min. 551 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 bu 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 a 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. A er derivatization with FMOC-Cl, the compounds become separable by reversed phase HPLC using an acetonitrile-phosphate mobile phase. e derivatization can be completed under mild conditions at 40 °C for 10 min; the deriva- tives are stable for at least 19 h. 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 552 Glufosinate and glyphosate e method with uorescence detection is higher in both speci city and sensitivity than that with UV detection. ere is another report [9] dealing with HPLC with 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 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 bu 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 so ware as described in the sec- tion of HPLC analysis with uorescence detection, 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); 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 bu er solution (pH 8.5) and 100 µL of 1 % PNBC acetonitrile solution, and le at 22–25 °C for 10 min; 10 µL of the solution is injected into HPLC. Assessment of the method 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 553 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 a er derivatization, their peaks appear at early retention times by the re- versed phase HPLC. 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. e λ max 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. 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. 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 554 Glufosinate and glyphosate Toxic and fatal concentrations GLUF [18] 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). e oral LD 50 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. e oral LD 50 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; a er this period, poisoning symptoms, such as lowering of the consciousness level, respiratory suppression and generalized convulsion, suddenly appear. e severity of the GLUF poisoning can be predicted by plotting the time a 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 a 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. 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 e ects in many GLUF poisoning cases. GLUF shows contradictory e ects on the central nervous system, viz., its excitation and suppression; it may act on glutamate synthase, glutamate decarboxylase and inhibitory glutamic acid receptors. 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 [...]... between aggravation of poisoning by intake of a glufosinate-containing herbicide (BASTA Fluid®) and serum glufosinate concentrations J Jpn Assoc Acute Med 8:617–618 (in Japanese) 4) Goto M, Kato M (1987) Method for Analysis of Residual Pesticides, Enlarged edn Soft Science, Tokyo, p 236 (in Japanese) 5) Kageura M, Hieda Y, Hara K et al (1988) Analysis of glyphosate and (aminomethyl) phosphonic acid in a. .. 18:305–308 20) Honda T, Nakashima K, Higashi H et al (1999) Analysis of toxicokinetics of glufosinate (GLF) in a case of poisoning by oral intake of BASTA Fluid® Jpn J Toxicol 12:468 (in Japanese) 21) Hori Y, Koyama K, Fujisawa M et al (2001) Protein binding of glufosinate and factors affecting it revealed by an equilibrium dialysis technique J Anal Toxicol 25:439–442 22) Japan Poison Information Center... glyphosate, glufosinate and bialaphos and their metabolites by capillary gas chromatography-ion-trap mass spectrometry J Chromatogr 637:167–173 16) Stalikas CD, Konidari CN (2001) Analytical methods to determine phosphonic and amino acid group-containing pesticides J Chromatogr A 907:1–19 17) Stalikas CD, Pilidis GA (2000) Development of a method for the simultaneous determination of phosphoric and amino... poisoning case Jpn J Legal Med 42:128–132 6) Tsunoda N (1994) Analysis of phosphorus-containing amino acid-type herbicides and their problems in forensic examination Jpn J Forensic Toxicol 12:104–107 (in Japanese with an English abstract) 7) Hori Y, Fujisawa M, Shimada K et al (2001) Determination of glufosinate ammonium and its metabolite 3-methylphosphinicopropionic acid in human serum by gas chromatography-mass... HPLC analysis with UV absorption detection 10) Akuzawa N, Akaiwa H (1997) Rapid determination of DL-homoalanin-4-yl-(methyl)phosphinic acid by highperformance liquid chromatography with fluorescence detection Bunseki Kagaku 46:69–74 (in Japanese with an English abstract) 11) Nishida K, Narihara M, Tsutsumi K et al (1998) A study on a screening method for water-soluble herbicides Abstracts of Annual Meeting...Toxic and fatal concentrations About 20 % of total GLUF, which had been orally administered to rats, is rapidly absorbed into the animal bodies; about 90 % of the absorbed GLUF is excreted into urine also rapidly In humans, a peak serum GLUF concentration is observed 40–50 min after ingestion; more than 95 % of an absorbed amount of the compound is excreted into urine within 24 h; a major part of the... Meeting of Japanese Association of Science and Technology for Identification, p 69 (in Japanese) 12) Sato K, Suetsugu K, Takegoshi Y et al (2000) Ion chromatography with electrochemical detection for phosphorus-containing amino acid type herbicides Abstracts of Annual Meeting of Japanese Association of Science and Technology for Identification, p 84 (in Japanese) 13) Honda M, Sato M, Kikuchi M et al... and finally a shock state due to reduced total blood volume In animal experiments, about 30 % of an orally administered amount GLYP is absorbed into bodies through the digestive tract, and a peak blood GLYP concentration can be attained in 3–4 h The main excretion route is the urinary system; but a part is excreted into feces Notes a) As an IS except APPA, N-(phosphonomethyl )- -alanine [13] is usable,... chromatography-mass spectrometry following mixedmode solid phase extraction and t-BDMS derivatization J Anal Toxicol 25:680–684 8) Suzuki A, Kawana M (1989) Rapid and simple method for identification of glufosinate- ammonium using paper chromatography Bull Environ Contam Toxicol 43:17–21 9) Okuda T, Naotsuka K, Sameshima I et al (1993) A new HPLC analysis of glufosinate caused acute poisoning Jpn J... 1) Tsunoda N (1990) Phosphorus-containing amino acid type herbicides Jpn J Forensic Toxicol 8:100–111 (in Japanese) 2) Koyama K, Hirose Y, Taze C et al (2000) An indicator of aggravation in poisoning by intake of BASTA Fluid®; comparison of estimated intake amounts with serum glufosinate concentrations ([GLF]s) Jpn J Toxicol 13:469 (in Japanese) 3) Koyama K, Hirose Y, Okuda T et al (1997) Relationship . HPLC-UV [14], a er each derivatization for analysis of PAAHs, are described. GC/MS analysis [7] Reagents and their preparation • GLUF (DL-homoalanin-4-yl(methyl)phosphinate. 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