Anal. Chem. 1982, 5 4 , 1082-1087
1082
confidential preprint describing their results on the analysis of leukotrienes. We thank Linda Missias for her assistance during the preparation of this manuscript.
LITERATURE CITED Samuelsson, 6.; Borgeat, P.; Hammarstrom, S.; Murphy, R. C. Prostaglandins 1979, 17, 785-787. Borgeat, P.; Samuelsson, B. Proc. Natl. Acad. Sci. U . S . A . 1979, 76, 3213-3217. Murphy, R. C.; Hammarstrom, S.; Samuelsson, B. Proc. Natl. Acad. Sci. U . S . A . 1979, 76, 4275-4279. Piper, P. “SRS-A and Leukotrienes”; Wiley: New York, 1981. Matthews, W. R.; Rokach, J.; Murphy, R. C. Anal. Blochem. 1981, 778, 96-101. Corey, E. J.; Clark, D.;Goto, G.; Marfat, A.; Mloskowski, C.; Samuelsson, 8.; Hammarstrom, S. J . Am. Chem. SOC. 1980, 102, 1438- 1439.
(7) Corey, E. J.; Clark, D.; Marfat, A,: Goto, G. Tetrahedron Lett. 1980, 21, 3143-3146. (8) Corey, E. J.; Albright, J.; Barton, A,; Hashlmoto, S. J . Am. Chem. SOC. 1980, 102, 1435-1436. (9) Bach, M.; Brashler, J.; Hammarstrom, S.; Samuelsson, B. J . Immuno/. 1980. 125. 115-117. (10) Karger, 6.; Martin, M.; Guichon, G. Anal. Chem. 1974, 4 6 , 1640-1 647. (11) Radmark, 0.; Malmsten, C.; Samuelsson, 6.; Goto, G.; Marfat, A.; Corey, E. J. J . Biol. Chem. 1980, 225, 11828-11830. (12) Karger, 6.; Giese, R. Anal. Chem. 1978, 50, 1048A-1073A. (13) Wynalda, M.; Llncoln, F.; Fitzpatrick, F. J . Chromatogr. 1979, 176, 4 13-4 17.
RECEIVED for review December 23,1981.
Accepted February
22, 1982.
Liquid Chromatographic Determination of Alkylthiols via Derivatization with 5,5’-Dithiobis(2=nitrobenzoic acid) Kazuhlro Kuwata, * Mlchlko Ueborl, Kazuhlko Yamada, and Yoshlakl Yamazakl Environmental Pollutlon Control Center, 62-3, 1 Chome, Nakamlchi, Hlgashlnari-ku, Osaka City 537, Japan
Traces of C1-C.I alkylthlols were derlvatlzed with 5,5’-dlthlobls(2-nltrobenzolc acld) In an aqueous medlum at pH 8, and the resulting dlsulfldes were determined by reversed-phase high-performance liquid chromatography. The analytical column used (20 cm X 4.8 mm 1.d.) was packed wlth LIChrosorb RP-18 (5 pm) and the moblle phases were 55% methanol/20 % phosphate buffer (pH 8)/25 % water for the Cl-C4 fhlols and 70% methanolR0% phosphate buffer/lO% water for the C,-C, thiols. Interferences from some possible metal Ions were mlnlmlzed by use of ethylenedlamlnetetraacetic acld. The detection llmlts of the thlols were 0.1-0.5 ng. The thiols In the range 0.2-25 pg/mL In aqueous media could be determined wlth 1.0-5.4% relatlve standard devlatlon. The thiol vapors In the low parts-per-mllllon or partsper-bllllon range could be dlrectly trapped by bubbling them Into the reaction medlum and determlned with less than 5.3 % relatlve standard devlation.
Low-molecular alkylthiols have received much attention as odors, flavors, or metabolites in environmental, food, and biological studies. Gas chromatography (GC) is widely used to determine traces of such alkylthiols (1-5). However, considerable difficulty is encountered in storing and analyzing alkylthiol samples especially in extremely low concentration levels because of the reactivity and their adsorption on solid surfaces. Traces of the alkylthiols often disappear even during the GC analysis. Hence, accurate determination of the alkylthiols by GC may be limited to more than 10 ng despite the sensitivity of a flame photometric detector. Additionally, the compounds of interest cannot be easily resolved from the interfering organic substances in complex samples. Certain disulfides, such as 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) (Ellman’s reagent) (6-15), 2,2‘-dithiodipyridine (15, 16), 2,2‘-dithiobis(5-nitropyridine)( I 7, 18), 4,4’-dithiodipyridine (15,19,20), and other disulfides (21))are frequently used for the spectrophotometric determination of thiol groups in a variety of biological samples. When a thiol compound
is allowed to react with the disulfide which is present in excess, a mixed disulfide and the corresponding thiol are formed as shown
RSH
+ R’SSR’
-
RSSR’ + R’SH
The reaction has been found to be rapid and quantitative at room temperature. As the corresponding thiol absorbs a t a different wavelength than the disulfide, the total amount of thiol groups has been successfully quantified by determining the absorbance of the thiol (6-21). Thiol compounds, however, have not been separately determined by this method. So far, there are few published methods for the determination of traces of alkylthiols, except for high-molecular alkylthiols (22), by high-performance liquid chromatography (HPLC). In this paper, a convenient method is presented to determine C1-C7 alkylthiols at low nanogram levels by reversed-phase HPLC via the derivatization with DTNB
as an ultraviolet (W)labeling agent. Stable standard samples were prepared for long-term use. Successful applications of the technique for determination of the alkylthiols a t trace levels in gas and vegetable samples are presented. EXPERIMENTAL SECTION Reagents and Materials. Methanethiol (98.5%) was purchased from Seitetsu Chemical Industry (Osaka,Japan), and the other alkylthiols used as standards were of special grade from Wako Pure Chemical Industries (Osaka, Japan). 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB) was from Dojindo Laboratories (Kumamoto, Japan), The other chemical reagents used were of special grade from Wako Pure Chemical Industries and Tokyo Kasei Kogyo (Tokyo, Japan). Methanol was of liquid chromatographic grade from Wako Pure Chemical Industries. The phosphate buffer (Phos) (pH 8) was 6.3 X M dipotassium phosphate and 3.3 X lo4 M potassium biphosphate in water. The Tris-glycine-ethylenediaminetetraacetic acid (EDTA) buffer M tris(hydroxylmethy1)amino(Tris-Gly) (pH 8) was 8.6 X
0003-2700/82/0354-1082$01.25/00 1982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
1083
-
Table I. Effects of Metal Ions on the Determination of the Alkylthiols concn of thiol,a dmL 2.08 3.25
Cd( 11)
Co( 11)
Cr(VI)
Cu( 11)
Fe( 111)
Pb( 11)
Zn( 11)
97.4 100.5
82.7 100.3
94.7 96.3
96.1 98.4
96.4 100.8
101.1
2-propene-1-thiol
3.04
100.0
97.8
100.0
98.8
101.2
92.9
94.9
96.8
2-propaneth io1
1.17
104.6
102.0
97.4
90.8
113.1
92.8
108.5
96.7
1-propanethiol
6.14
102.0
100.9
99.9
94.7
105.4
98.3
101.1
101.’7
2-methyl-2-propanethiol
8.73
93.1
99.1
97.5
91.0
101.2
96.2
99.7
99.7
2-butanethiol
3.49
110.1
105.1
97.0
94.9
116.5
93.7
108.0
97.6
101.3
99.8
97.6
99.1 30.0 (99.5) 24.1 (80.8) 15.0 (69.9) 30.9 (98.9) 47.3 (99.0) 13.9 (75.1) 27.9 (93.3) 14.1 (96.3) 11.5 (77.8) 8.4 (65.8) 31.3 (51.7)
I W 11) 104.2 103.5
Ni( TI)
99.5 100.0
92.7
107.6
97.8
100.5
99.8
92.7
111.0
94.2
105.8
95.3
98.4 (98.2) 51.1 (91.8) 12.6 (78.3)
96.9
106.6
100.7
103.7
106.1
97.8
94.4 (98.4:) 87.8 (89.0) 73.9 (85.7)
thiol methane thiol ethane thiol
2-methyl-1-propanethiol
10.0
retained a b s o r b a n q b %, for the metal ionC
1-butanethiol
3.14
115.2
100.5
97.9
1-pentanethiol
6.75 8.31
1-heptanethiol
7.43
108.4 (100.4) 82.7 (97.2) 51.3 (88.7)
97.8
1-hexanethiol
63.4 (100.9) 18.7 (67.7) 6.5 (29.6)
101.6 101.7
105.8 101.2 97.8
98.0
Percent absorbance retained in the metal-added sample; absorbance was a Concentration of thiol in the final medium. Metal ion concentration at 20 Hg/mL and at 5 pg/mL in parendetermined from peak height by using 2 9L of the medium. A methanethiol vapor was bubbled through 10 mL of the sampling solution A. theses. .
M EDTA in water M glycine, and 2 X methane, 9.2 X (14). The DTNB solution was 0.5% DTNB in Phos. The sampling solution A was 0.3% DTNB in 90% Phos/lO% Tleis-Gly. The sampling solution B was 0.3% DTNB in 80% Phos/lO% Tris-Gly/lO% 2-butanol. The analytical column was a 20 cm X 4.6 mm i.d. stainless steel tube slurry-packed with LiChrosorb RP-18 (5 bm) (E. Merk, Darmstadt, West Germany) (23). Apparatus. A Water Associates (Milford, MA) ALC/GPC 244 liquid (chromatograph equipped with a U6K injector and a JASCO (Tokyo, Japan) UVIDEC 100 I11 UV absorbance detector adjusted to 330 nni was employed. The mobile phases were 55% methanol/2O% Phos/25% water for the C1-CI alkylthiols and 70% methmol/20% Phos/lO% water for the C,-C, alkylthiols, and the flow rate was 1.0 mL/min. Preparation of the Standard Solutions. For methanethiol standards, 20 mL of methanethiol gas (98.5%) was taken at 20 OC in a calibrated 1000-mL gastight glass tube under reduced pressure of nitrogen by using a gastight syringe, and nitrogen was introduced into the tube up to 1atm. Then, 30 mL of the diluted sample (2% methanethiol) in a volumetric syringe was introduced into a stream of 0.5 L/min nitrogen and bubbled with two fritted bubblers (60 mL) in series with 10 mL of the sampling solution A. The solutions were combined and brought up to 50 mL with Phos. For the C2-C7alkylthiols, 0.2-0.3 g of the thiols was dissolved in 50 mL of 50% 2-propanol in 0.2% potassium hydroxide solution. The solutions of the individual alkylthiols were diluted by a factor of 10 with 50% 2-propanol/50% Phos. Five rniUiliters of the diluted solutions was taken in a 50-mL volumetric flask, and 5 mL of Tris-Gly and 20 mL of the DTNB solution were added. The mixture was brought up to 50 mL with Phos and used as a stock sollution. Standards of lower concentrations were made by appropriately diluting the stock solution of each thiol with Phos. The stock solutions were stored in tightly closed conlahers at 3 O C in the dark. Preparation of Vegetable Sample. Ten to twenty grams of a sample piece (garlic or onion) was homogenized with 20 mL of distilled water in a homogenizer. The sample was brought up to 50 mL with distilled water. A part of the sample was centrifuged for analysis of the supernatant. Analytical Procedure. Aqueous Sample. One to six milliliters of a sample was mixed with l mL of Tris-Gly and 2 mL of the DTNB solution, and the mixture was brought up to 10 mL with Phos (pH 8). Then, 2-10 ELLof the aqueous sample was analyzed by IHPLC. The identification of the alkylthiols was made
by retention time, and the quantitation was performed by peak height. Gas Sample. A volume of 1-150 L of a gas sample was bubbled at 0.5-1.5 L/min through two fritted bubblers (60 mL) in series with 10 mL of the sampling solution A or B (pH 8). For tobacco smoke, 140 mL of smoke from four puffs was filtered with a Toyo Roshi (Tokyo,Japan) No. 5A paper filter to filter particulate arid tar substances, and the filtered substance was bubbled with a carrier of 0.5 L/min of clean air (24, 25). The solutions were combined,brought up ID 30 mL with Phos, and analyzed by HPLC as described above. Vapor from Vegetable Sample. Thirty milliliters of a homogenized sample was placed in a 300-mL flask and stirred. Nitrogen was passed on the surface of the sample at 60 mL/min for 8-24 h and bubbled through two fritted bubblers with 10 mL of the sampling solution A. The sampling solutions were combined, brought up to 30 mL with Phos, and analyzed by HPLC as described above.
RESULTS AND DISCUSSION Twelve C1-C7 alkylthiols were derivatized with DTNB in weak alkaline solution and the derivatized disulfides deteipmined by HPLC. DTNB was chosen as a derivatization reagent because of its high water solubility. The DTNB solution was usable for at least a week because nitromercaptobenzoic acid, a decomposition product of DTNB, did not interfere with the determination of the alkylthiols. The reaction of the alkylthiols with DTNB appeared to proceed rapidly and quantitatively in the range pH 7-9 in the presence of excess DTNB as described elsewhere (6, 12, 14, 15, 18). The pH, however, should be adjusted to 8 so as t o minimize effecb of coexisting metal ions in the reaction. There are some metal ions that could form mercaptides with thiol compounds or oxidize thiol compounds thus inhibiting the reaction of the thiols with DTNB. Use of EDTA in the reaction medium adjusted to pH 8 was effective in reducing such interferences. Table I indicates the effects of various metal ions a t levels of 5 and 20 pg/mL. In the case of methanethiol, 2 L of a synthetic air samples was bubbled through the sampling solution A containing 20 pg/mL of a metal ion. For the other thiols, EDFA was added to a mixture of an alkylthiol and a metal ion prior to addition of the DTNB solution. The
1084
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
Table III. Stability of the DTNB Derivatives of the Alkylthiols concn of thiol: pg/mL
thiol methanethiol e thanethiol
250
300 350 Wavelength, n m
2-propene-1-thiol 2-propanethiol 1-propanethiol 2-methyl- 2-propanethiol 2-bu tanethiol 2-methyl-I-propanethiol 1-butanethiol 1-pentanethiol 1-hexanethiol 1-heptanethiol
LOO
Figure 1. Absorption spectra 5f -!he reaction products: sample lnjected, 3-11 ng as thiols in 2 pLL; (1) DTNB, (2) methanethiol derivatlve (dew),(3) ethanethiol derv, (4) 1-propanefhiolderv, (5) I-butanethiol derv, (6) nitromercaptobenzoic acid.
3.88 5.56 5.57 7.31 11.1
16.4 14.0 14.9 14.8 13.1 11.3
13.0
retained abs,b % 83.2 92.3 87.7 94.9 95.0 98.9 96.6 96.3 97.2 100.1 97.9 95.5
a Concentration of thiol in the final medium. Percent absorbance retained after 20 days of storage at room temperature (20 "C) without protection against light.
Table 11. Extinction Coefficients for the DTNB Derivatives of the Alkylthiols at 330 nm a '
thiol
€330,
M-I
cm"
thiol
E 330
M - l cm-'
methanethiol 8900 2-butanethiol 9000 e thane thiol 8940 2-methyl-l8530 2-propene86 50 propanethiol 1-thiol 1-butanethiol 8520 2-propanethiol 8980 1-pentanethiol 8480 1-propanethiol 8720 1-hexanethiol 8510 2-methyl-28940 1-heptanethiol 8700 propanethiol average 8740 t 203 (standard deviation) a Extinction coefficient (€330nm)of each derivative was calculated from the separate peak in the chromatogram as follows: E330nm = ( A ~ / L , ) ( M , / m ) W , , , R , where Ah was absorbance in peak height, L , was pathlength of the cell (1.0 cm), M , was molecular weight of a thiol (g), m was amount of the thiol injected (g), W , , , was peak width at half-height (min), and R was flow rate of the mobile phase (0.001 L/min).
concentration of EDTA was 2 X lo4 M. Cu(I1) at a level of 20 pg/mL gave a significant decrease in absorbance for C& thiols. The effects were not appreciably reduced by increased use of EDTA up to 2 X M. Significant effects of Cd(II), Co(II), Hg(II), and Zn(I1) were observed at a level of 20 pg/mL only on the determination of C6-C7 thiols. However, the effects of these metal ions were small, except Cd(II), with less than 2-5 pg/mL levels in the medium. When the alkylthiols were added to the reaction medium at a metal level of 20 M EDTA, little interferences were obpg/mL with 2 X served on the determination of the alkylthiols. Reducing impurities in the sample, such as thiocyanate, cyanide, thiosulfate, sulfide, or sulfur dioxide, did not interfere with the determination in the presence of excess DTNB. The UV spectra of the DTNB derivatives of the alkylthiols were obtained by determining the absorbances a t every 5 nm in the range of 200-400 nm after separating the derivatives by HPLC. Figure 1 shows that the DTNB derivatives gave maximum absorbances around 330 nm, and Table I1 reports that the averaged extinction coefficient for the DTNB derivatives at 330 nm was 8740 M-l cm-l with a standard deviation of 203 M-l cm-l. The DTNB derivatives were quite stable in the presence of excess DTNB. Table I11 reports that the DTNB derivatives in the standard samples were stable for at least 3 weeks a t room temperature (20 "C) without protective covers against light. When the sample solutions
0
5
10 15 20 Relention t ime. min
25
Figure 2. Retention times of DTNB derivatives vs. methanol content in the mobile phase: flow rate, 1.0 mL/min; DTNB derivative of (1) methanethlol, (2) ethanethlol, (3) I-propanethiol, (4) 1-butanethiol, (5) 1-pentanethiol,(6) I-hexanethiol, (7) I-heptanethioi.
were stored in tightly closed containers at 3 "C in the dark, the DTNB derivatives were stable for as long as a few months. Thus, standard solutions and sample solutions after the derivatization may be usable for analysis over a considerably long period, The analytical column packed with LiChrosorb RP-18 (5 hm) offered 12000 theoretical plates (16.7 pm HETP) for ethanethiol. Addition of the phosphate buffer to the mobile phase was required to separate the DTNB derivatives of the individual alkylthiols from DTNB and nitromercaptobenzoic acid. Figure 2 shows that the retention times of the DTNB derivatives could be controlled by changing the methanol content in the mobile phases. For determination of higher alkylthiols such as the C5-C7thiols, use of a mobile phase with a higher methanol content was recommended for increasing analytical sensitivity and for decreasing analysis time. The mobile phases used for routine analysis were 55% methano1/20% Phos/25% water for the C1-CI thiols and 70% methanol/20% Phos/lO% water for the C5-C7 thiols. Table IV reports the capacity factors ( k 3 of the DTNB derivatives of the alkylthiols in the HPLC system. The interday relative standard deviation of the k'was less than 0.6%. Figure 3 shows typical liquid chromatograms of the DTNB derivatives under the above conditions. Excellent separations were obtained for the C1-C7 thiols, especially for the C3-C4 isomers. A gradient elution programmed in the range 30-80% of methanol content in the mobile phase may be also effective in separating simultaneously the Cl-C7 thiols though a much longer analysis time is required in repeated runs.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
10@15
Table I V . Capacity Factors ( k ' )of the DTNB Derivatives of the Alkylthiols in the HPLC System
k' thiol methianethiol ethanethiol 2-propene-:l-thiol 2-propanethiol 1-propanethiol 2-methyl-2.propanethiol 2-butanethiol 2-methyl-1.propanethiol 1-butanethiol 1-pentanethiol 1-hexanethiol 1-heptanethiol
1.02 1.73 2.08 2.92 3.21 4.49 5.23 5.67 6.06 11.9 23.9
10
1:
2.43 3.99 6.58
a A: mobile phase, 55% methanol/20% Phos/25% water. El: mobile phase, 70% methanol/20% Phos/ 10% water.
Table V. Determination Ranges of the Alkylthiolsa
thiol methanethiol ethanethiol 2-propene- 1thiol 2-propanethiol 1-wopanethiol 2-methyl-2. propanethiol 2-butanethiol 2-met hy 1-1pro panet hiol 1-butanethiol 1-pentanethiol 1-hexanethiol 1-heptanethiol
detection limit, ng
0
linear range,b - rg/mL min m ax
5 10 15 Retention time, m i n
20
25
0.1 0.2 0.2
0.2 0.3 0.3
35 45 45
0.3
0.6
45
Flgure 3. Liquid chromatograms of the DTNB derivatives of the alkylthlols. Conditions: mobile phase, (A) 55 % methanoV20 % Phos/25 % water, (B) 70 % methanol/20 % Phos/lO % water; flow rate, 1.0 mL/min; sample injected, 5-40 ng as thiols in 2 pL. Peak identity: (1) methanethiol, (2) ethanethiol, (3) 2-propene-1-thiol, (4) 2-propanethlo1, (5) 1-propanethiol, (6) 2-methyl-2-propanethio1, (7) 2-butanethiol, (8) 2-methyl-1-propanethiol, (9) 1-butanethiol, (10) 1-
0.3
0.6
45
pentanethiol, (1 1) 1-hexanethiol, (12) I-heptanethiol, (13) nitromercaptobenzoic acid, (14) DTNB.
0.5
1.0
50
0.5 0.5
1.0 1.0
50 50
0.5 0.3 0.4 0.5
1.0 0.5 0.6 1.0
50 30 50 50
alkylthiols were observed in the blank tests with distilled wateir and 150 L of air purified with molecular sieve 13X for gas samples. Table V reports the detection limits (a response to twice the noise level) and the linear determination ranges for the alkylthiols. The detection limits might be improved to some extent by shortening the retention times, if required. The estimated detection limits of the alkylthiols in vapor. phase were 1-3 ppb (v/v) for 150 L of a gas sample a t 25 "C. The upper limits in the determination ranges were dependent, on the linear range of the instrument, the concentration of DTNB, and the solubility of the alkylthiols in the reaction1 medium. In conventional GC methods ( I d ) , alkylthiols at low nanograms are difficult tx) determine with a reasonable precision despite use of a flame photometric detector because of their adsorption on the injection port, the column packing, and/or column tubings. In this HPLC method, the alkylthiols at less than 1ng could be accurately determined because of the low
a Analytical conditions: see Figure 3. Concentration of alkvlthiol iin the final medium usinn 2 vL.
Interfering organic compounds were not present in most cases because few organic peaks that overlapped with those of the DTNB derivatives of alkylthiols were found a t 330 nm. If interfering organic peaks were present, washing of the sample with a small volume of chloroform or carbon tetrachloride was used to remove the organics since the DTNB derivatives were not extracted into the organic layer. No
Table VI. Determination of the Alkylthiols in Synthetic Air Samples concn of thiola thiol methanethiol ethanethiol 2-propene-1-thiol 2-propanethiol 1-propanethiol 2-methyl-2-lpropanethiol
sample 1
-.
std dev, ppm (v/v) (collection efficiency, %)
sample 2cpd
0.206 ?: 0.0042 (98.0) 0.634 i 0.0148 (96.0) 1.98 i 0.0437 (97.0) 0.334 i. 0.0158 (88.9) 0.587 j: 0.0221 (93.8) 0.512 ?: 0.0273 (80.6) 0.562 i 0.0152 (86.7) 0.660 + 0.0230 (87.9) 0.511 i 0.0219 (90.1)
2-butanethiol 2-methyl-1-propanethiol 1-butanethiol 1-pentanethiol 1-hexanethiol 1-heptanethiol a Average concentration of thiol in six runs. used. e Sampling solution B used.
i
sample 3
sample 4 c , e
3.76 i 0.078 (98.5) 24.0 f 0.56 (96.3) 10.4 + 0.21 (98.7) 11.8 i 0.56 (88.1) 19.3 2 0.67 (93.4) 14.4 i 0.56 (75.6) 17.6 f 0.70 (84.5) 19.6 i 0.83 (85.9) 18.3 i 0.72 (90.2) 1.17 i 0.056 (90.1) 1.55 i 0.052 (90.4) 1.73 i 0.048 (88.3)
Sample volume, 8 L.
Sample volume, 2 L.
8.75 ?: 0.417 (89.5) 10.6 i 0.51 (90.7) 11.0 f 0.30 (92.8)
Sampling solution A
1086
ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982
Table VII. Alkylthiols in Gas and Vegetable Samples methanethiol
sample domestic fuel gas,u ppm (v/v) tobacco smoke, p g/cigarette Hi-light (Japan) Seven-star (Japan) Cherry (Japan) Peace (Japan) emission gas from a fermentation system of pig wastes, ppm (v/v) solubilizer fermenter vegetable sources garlic, supernatant, pg/g garlic, vapor emitted,e ng/g.h onion, supernatant, fi /g onion, vapor emitted, ng/g.h
9
Sample volume, 6 L. Average value in three runs. Sampling time, 24 h.
8 h.
I
l-propanethiol
0.13 280 315 364 368 0.13 0.18
1.24
3880 44.7 67.5 1.75 5.04 Sample volume, 10 L. Sample volume, 2 L. 316 11.8 2.44 4.21
20.5 3.25 e
Sampling time,
vapors, use of the sampling solution B was recommended for increasing the collection efficiency. Table VI reports that the alkylthiol vapors a t the low parts-per-million or parts-perbillion levels could be satisfactorily collected in the first two bubblers and determined with 2.0-5.3 % relative standard deviation. Alkylthiols in domestic fuel gas, tobacco smoke, and effluent gas of a methane fermentation system and in garlic and onion were determined. Table VI1 reports analytical data of these samples, and Figure 4 shows typical liquid chromatograms of the alkylthiols from the sources. Traces of the alkylthiols in the various samples were successfully determined. The HPLC method proposed may have advantages over conventional methods in terms of easy separation of the individual alkylthiols, high analytical sensitivity, low background effects, and stability of samples. The absorbance values for the alkylthiols are linear and reproducible over reasonably large ranges of concentrations. Determination of the alkylthiols by use of the proposed method could probably be applicable to many other types of samples after necessary modifications are made as required.
Il /
0
thiol found 2-propene2-propane1-th io1 thiol
1
I
I
I
10 15 20 Retention t i m e , min
5
Flgure 4. Analysis of alkylthlols in gas and vegetable samples: (A) tobacco smoke, (1) methanethlol (2.8 ng), 350 pg/cigarette; (6) emission gas from a methane fermenter, (1) methanethlol (0.22 ng) 0.18 ppm (v/v), (2) ethanethiol (less than the detection limit), (3) 2propene-1-thlol(0.99 ng), 1.24 ppm; (GI) garlic, supernatant undiluted, (1) methanethlol, (3) 2-propene-1-thiol, (4) 2-propanethiol (10.2 ng), 44.7 pg/g, (5) 1-propanethiol(4.08 ng), 20.5 pglg; (G2) garlic, supernatant dlluted by 10 tlmes with Phos, (1) methanethiol (7.55 ng), 316 pg/g, (3) 2-propene-1-thiol (88.7ng), 3880 pg/g, (4) 2-propanethlol; (5) I-propanethbl; (0) garlic, vapor emitted from the homogenized sample, (1) methanethiol (0.55 ng), 11.8 ng/g-h, (3) 2-propene-1-thiol (3.21 ng), 67.5 ng/g-h.
adsorption of the derivatization products. The C1-C7 alkylthiols in the aqueous media could be determined at 0.2-25 pg/mL levels with 1.&5.4% relative standard deviation. The standard deviation near the detection limits was 12-15%. For investigation on the collection efficiency in the sampling system and on the accuracy of analysis of gas sample, 60 L of air samples containing 0.2-24 ppm (v/v) of the alkylthiol vapors were prepared in polyester bags. A volume of 2-8 L of the synthetic samples was sampled at 25 O C five times through three fritted bubblers in series with 10 mL of the sampling solution A or B. In the sampling of the C6-C7 thiol
ACKNOWLEDGMENT The authors cordially thank K. Negoro, Faculty of Technology, Hiroshima University, for his continuous encouragement and advice in the study.
LITERATURE CITED (1) Stevens, R. K.; Mulik, J. D.;O'Keefe, A. E.; Krost, K. J. Anal. Chem. 1971. 4 3 . 827-831. (2) Bruner, F.'; Liverti, A.; Passanzini, M.; Allegrini, I. Anal. Chem. 1972, 4 4 , 2070-2074. (3) Pecsar, R . E.; Hartman, C. H. J. Chromatogr. Sci. 1973, 1 1 , 492-502. (4) Hoshika, Y.: Kozlma, I.; Koike, K.; Yoshimoto, K. Bunseki Kagaku 1974, 2 3 , 1393-1398. (5) Blanchette, A. R.; Cooper, A. D. Anal. Chem. 1976, 4 8 , 729-731. (6) Ellman, G. L. Arch. Biochem. Siophys. 1959, 8 2 , 70-77. (7) Butterworth, P. H. W.; Baum, H.; Porter, J. W. Arch. Biochem. Biophys. 1967, 118, 716-723. (8) Grassettl, D. R.; Murray, J. F., Jr. Arch. Biochem. Biophys. 1967, 119, 41-49. (9) . . Robvt. J. F.: Ackerman, R. J.; Chlttenden, C. G. Arch. Biochem. Biophys. 1971, 147, 262-269. (10) Sedlak, J.; Lindsay, R. H. Anal. Biochem. 1968, 2 5 , 192-205. (11) Koka, M.; Mikoijcik. E. M.; Gould, I. A. J. Dalry Sci. 1968, 51, 2 17-219. (12) Kalab, M. J . Dairy Sci. 1970, 5 3 , 711-718. (13) Glaser, C. B.; Maeda, H.; Meienhofer, J. J. Chromatogr. 1970, 5 0 , 151-154. (14) Beveridge, T.; Toma, S. J.; Nakai, S. J . Food Sci. 1974, 3 9 , 49-51. (15) Gabor, G.; Vlncze, A. Anal. Chim. Acta 1977, 9 2 , 429-431. (16) Grassetti, D.R.; Murray, J. F., Jr. Anal. Biochem. 1967, 2 1 , 427-434. (17) Grassettl, D.R.; Murray, J. F., Jr. J . Chromatogr. 1969, 41. 121-123. (18) Swatditat, A.; Tsen, C. C. Anal. Biochem. 1972, 4 5 , 349-356.
Anal. Chem. 1982, 5 4 , 1087-1090 (19) Ampulski, R. S.; Ayers, V. E.; Morell. S. A. Anal. Biochem. 1969, 32, 183-1 69. (20) Taketa, F.; Morell, S. A. Anal. Biochem. 1969, 32, 169-174. (21) Novak, T. J.; Pieva, S. G.; Epstein, J. A n d . Chem. 1980, 52, 1851-1055. (22) Cox, J. A.; Przyjazny, A. Anal. Lett. 1977, 10, 869-085. (23) Kuwata, K.; Uebori, M.; Yamazaki, Y. J . Chromatogr. 1981, 277, 370-302.
1087
(24) Kuwata, K.; Uebori, M.; Yamazaki, Y . J . Chromatogr. Sci. 1979, 17, 264-268. (25) Kuwata, K.; Uebori, M.; Yamazaki, Y . Anal. Chem. 1981, 5 3 , 153 1-1 534.
RECEIVED for review December
8,
Accepted February
22. 1982.
Liquid Chromatographic Determination of Guanadrel in Laboratory Animal Diet as the Fluorescent Acetylacetone Derivative Paul A. Bombardt and Wade J. Adams' Physical and Analytical Chemistry-Drug
Metabolism Research, The Upjohn Company, Kalamazoo, Michigan 4900 1
A hlgh-performance llquld chromatographic method for the rapld determlnatlon of guanadrel In laboratory anlmal dlet Is descrlbed. Derlvatlzatlon of guanadrel wlth acetylacetone In a homogeneous aqueous:organlc solvent permitted the sensltlve and speclflc fluorescence detection of the resuttlng pyrlmldlne wlthout the need for extractlon of the derlvatlve prior to chromatographic analysls. Wlth an excttatlon wavelength of 238 nm, the fluorescence response at 360 nm was linear for drug-dlet mlxtures havlng guanadrel sulfate concentratlons up to 1400 ppm and the llmlt of detectlon was approxlmately 1 ppm (3 ng oncolumn). Assay precision, as estlmated by analyzlng repllcate samples of a laboratory standard, was better than 1.5 % relative standard devlatlon.
The utility of derivatizing guanidino compounds with hexafluoroacetylacetone (1-5) or acetylacetone (6) to form the more volatile corresponding pyrimidines prior to gas chromatographic analysis is well documented. Quantitation of the pyrimidines using highly specific and sensitive electron capture or mass spectrometric detection has afforded sensitivities in the nanogram per milliliter range for guanidino compounds in plasma (1-3, 5 ) , urine (3),and tissue (4, 6). Analytical methods based upon the reaction of the amidino moiety with 8-hydroxyquinoline (Sakaguchi reaction) or phenanthrenequinone prior to colorimetric (7-9) or fluorometric (10, 11) detection, respectively, have also been reported. Separation of the guanidino compounds prior t o derivatization is necessary using these methods since these reagents do not yield unique derivatives. We report a rapid and specific reversed-phase liquid chromatographic method for the determination of guanadrel. Guanadrel sulfate is a guanidine antihypertensive agent currently under clinical investigation (12). The methodology was used for the quantitation of guanadrel in pelleted drugdiet mixtures administered to mice in a carcinogenicity study in order to comply with the FDA's Good Laboratory Practice regulations (13). Precolumn derivatization of guanadrel with acetylacetone in a homogeneous aqueous:organic solvent permitted the sensitive and specific fluorescence detection of the resulting pyrimidine without the need for extraction prior to chromatography.
EXPERIMENTAL SECTION Reagents. The reference standard guanidino compounds, guanadrel sulfate (I, [ (1,4-dioxaspiro[4.5]dec-2-yl)methyl]guanidine sulfate) and (cyclohexy1methyl)guanidinesulfate (II), were supplied by the Pharmaceutical Research and Development Laboratories of The Upjohn Company. Acetylacetone was obtained NH I1 CHz-NH-C-NH2 * % H2S04
5
NH
~ C H ~ - N H- -H L~ SNO H ~ ~ I/,
I
n
commercially (Eastman, Rochester, NY, 98% minimum purity; or Aldrich, Milwaukee, WI, 99%+ purity) and used without further purification. Distilled-in-glass spectroscopic grade acetonitrile, methanol and tetrahydrofuran (Burdick and Jackson, Muskegon, MI) were used as received. Inorganic chemicals were analytical reagent grade and were prepared in distilled, deionized water. Apparatus. The high-performance liquid chromatograph used in this study was a modular component system consisting of an Altex Model llOA solvent pump, an in-house designed and fabricated autoinjector (14) fitted with a 40-wL sample loop, a commerciallyprepared 4.6 rnm i.d. X 250 rnm column packed with 10-wm LiChrosorb RP-8 (E. Merck Laboratories, Elmsford, NY), a dual monochromator spectrofluorometer equipped with a 2 8 4 , flow cell (Model 2 W , Perkin-Elmer, Norwalk, CT), and a variable sensitivity recorder (Model 355,Linear Instruments, Irvine, CA). Automated data acquisition and processing were accomplished using an IBM 1800 computer (15). A two-speed reciprocating shaker (Eberbach and Sons, Ann Arbor, MI) was used for extraction of samples and a block heater (Lab-Line Instruments, Melrose Park, IL) was used for the derivatizations. Mass spectral characterization of the derivatives was accomplished by using a magnetic sector mass spectrometer (Model CH7A, Varian Mat, Bremen, West Germany). Sample Analysis. Internal standard and reference standard solutions containing approximately 700, 210, and 70 pg/mL of the respective compounds were prepared each day samples were analyzed by dissolving the accurately weighed reference standards in 0.05 M ammonium dihydrogen phosphate. Individual pellets of the diet were pulverized using a glass mortar and pestle and 1-g samples weighed into 16 x 125 mm culture tubes fitted with Teflon-lined caps. The standards were prepared by using drug-free pelleted diet. Following addition of 5 mL of 0.05 M ammonium dihydrogn phosphate, the samples were vortexed to expel trapped air and allowed to disintegrate for 15 min. One milliliter of the appropriate internal standard
0003-2700/82/0354-1007$01.25/00 1982 American Chemical Society
