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Enhancement and Stabilization of Dithizone Vital Staining for Zinc in Rat Organs Using Adduct Formation
NOTES & TIPS
was able to detect some other HA-binding proteins of high molecular weights also (Fig. 4C). It is also important to mention that the signal obtained by radioiodinated adduct is much more prominent compared to immunodetection, suggesting the high sensitivity of this method. The clinical importance of HA and its binding proteins is gaining importance due to its involvement in various diseases (1). The present method has advantages over the immunodetection method because it can detect a wide range of HA-binding proteins. In this regard, the applicability of this radioactive probe increases substantially and, consequently, it may have much wider applications in the future. REFERENCES 1. Knudson, C. B., and Knudson, W. (1993) FASEB J. 7, 1233– 1241. 2. Raja, R. H., Leboeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168–177. 3. Cardin, A. D., Witt, K. R., and Jackson, R. L. (1984) Anal. Biochem. 137, 368–373. 4. Yang, B., Hall, C. L., Yang, B. L., Savani, R. C., and Turley, E. A. (1994) J. Cell. Biochem. 56, 455–468. 5. Yu, Q., and Toole, B. P. (1995) Biotechniques 19, 122–129. 6. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849–857. 7. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330–334. 8. Laemmli, U. K. (1970) Nature 227, 680–685. 9. Gupta, S., Babu, B. R., and Datta, K. (1991) Eur. J. Cell. Biol. 56, 58–67. 10. Tengblad, A. (1979) Biochim. Biophys. Acta 578, 281–289. 11. Deb, T. B., and Datta, K. (1995) J. Biol. Chem., in press.
Enhancement and Stabilization of Dithizone Vital Staining for Zinc in Rat Organs Using Adduct Formation Chikako Shima, Kin-ichi Tsunoda, Hideo Akaiwa,1 Keiji Suzuki,* and Katsuyuki Nakajima† Department of Chemistry, Gunma University, Kiryu 376, Japan; *Department of Pathology, Medical Care and Technology, Gunma University, Maebashi 371, Japan; and †Japan Immunoresearch Laboratories Co., Ltd., Takasaki 370, Japan Received September 5, 1995
Zinc is one of the essential elements for mammalian species including human; numerous zinc enzymes have 1
To whom correspondence should be addressed. Fax: 81-277-301251. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
been found during the past several decades. Thus, the distribution of zinc in mammalian bodies has attracted wide attention in biomedical fields to elucidate its physiological role and metabolism. Histochemical staining techniques have often been used for such purposes (1). For example, the existence of zinc in the islet of Langerhans was shown by a dithizone staining method for zinc(II) (2), which has still been one of the most important staining methods for zinc in spite of some shortcomings such as its relatively low sensitivity (1–5). We have also applied the dithizone method to elucidate the zinc distribution in rat tissues. However, the ordinary staining method was not sensitive enough to detect zinc in a normal rat, even in tissues such as pancreas and prostate which are known to concentrate zinc (1). Thus, the vital staining method was applied to this problem (2– 5). Although violet color development due to the formation of zinc(II)–dithizonate was observed in several organs by this method, the positive staining faded out almost completely half a day after the specimen preparation because of the decomposition of the complex. In this paper, we overcome this problem by establishing an improved dithizone vital staining method in which adduct formation of zinc(II)–dithizonate with pyridine or 4-methyl pyridine was applied to stabilize the complex. With the present method, the positive staining was strongly enhanced and the period of the stable positivity was extended up to 7 to 8 days. All chemical reagents used in this study were analytical grade and used without further purification. Dithizone stain solution A. Dithizone (100 mg) was dissolved in 1 ml ethanol with 0.2 ml concentrated ammonia solution. Then the solution was diluted with 20 ml deionized water. Dithizone stain solution B. Pyridine base, i.e., pyridine or 4-methyl pyridine whose concentration was equivalent to or five times as high as dithizone, was added to the solution A. Dithizone vital staining of rat tissues for zinc (4–6). Male rats weighing ca. 200 g were used for all experiments. The dithizone stain solution A or B (3–4 ml) was infused by intraperitoneal injection into a rat anesthetized with ether. After 30 min, the organs were taken out, frozen with liquid nitrogen, then cut at a thickness of 3 mm with a cryostat. The sliced tissues were stained with methyl green and enclosed with a glycerol and gelatin mixture on a glass slide. The examined organs were brain, salivary gland, lung, thymus, heart, spleen, liver, pancreas, kidney, intestine, testis, and prostate. Among them, the violet color development due to zinc(II)–dithizonate complex was found in the pancreas (the islet of Langerhans), prostate (epithelial cells of lateral lobe), and intestine (Paneth’s cells) with the ordinary dithizone vital staining method (2–5). Figure 1 is a photomicrograph of prostate as an example. Because
236, 173–175 (1996)
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NOTES & TIPS
FIG. 1. Photomicrograph of rat prostate with dithizone vital staining (immediately after specimen preparation). No pyridine base was added to the stain solution. Epithelial cells are positive. Original magnification 1100.
of the decomposition of the complex, however, the violet color faded out almost completely half a day after the specimen preparation (Fig. 2). Thus, we tried to stabilize the Zn(II)–dithizonate and to prolong the positivity by the adduct formation with pyridine bases; the residual water molecules which coordinate to the central metal ion in the complex are replaced by the pyridine base, thus resulting in higher stability of the complex due to the prevention of oxidation reaction as well as the increase in its hydrophobicity (6). In this study, pyridine and 4-methyl pyridine were chosen as the pyridine base, because they have often been used as an adduct formation agent in solvent extraction of metal ions (7, 8). In general, 4-methyl pyridine is a better adduct-forming agent than pyridine because of its stronger basicity (6). At first, the sliced vital stained tissue was dipped in these pyridine base solutions. However, no
FIG. 2. Photomicrograph of rat prostate with dithizone vital staining (3 days after specimen preparation, the same specimen as that of Fig. 1). Positive staining of epithelial cells faded out gradually. Original magnification 1100.
/ 6m13$$$nat
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FIG. 3. Photomicrograph of rat prostate with dithizone vital staining (immediately after specimen preparation). 4-Methyl pyridine was added to the stain solution at the concentration equivalent to that of dithizone. Epithelial cells are strongly positive. Original magnification 1400.
effect was observed on the prevention of the positive staining from fading in this in vitro experiment. Thus, pyridine bases were added to the dithizone stain solution and injected intraperitoneally into the rat together with dithizone. The concentration of each base was the same or five times as high as that of dithizone. A remarkable enhancement of and stabilizing effect on the positive staining was observed in every case. The effects were more remarkable with 4-methyl pyridine than with pyridine as expected from their adduct-forming abilities. With the addition of the equivalent concentration of 4-methyl pyridine to dithizone, the stable positivity of zinc(II)–dithizonate lasted for up to 6 days. Figures 3 and 4 are photomicrographs of the vital stained rat prostate with dithizone and 4-methyl pyridine (equivalent concentration to dithizone), immediately after specimen preparation and after 6 days, respec-
FIG. 4. Photomicrograph of rat prostate with dithizone vital staining (6 days after specimen preparation, the same specimen as Fig. 4). Original magnification 1400.
AP-Anal Bio
NOTES & TIPS
tively. The dark violet granules can be observed in these figures. Essentially, the degree of the positive staining was much greater in Figs. 3 and 4 than in Fig. 1. The effect of 4-methyl pyridine on the prevention of the color from fading (Fig. 4) was also remarkable compared with Fig. 2. Moreover, with the addition of a five times higher concentration of 4-methyl pyridine, the positivity was further strongly enhanced and the period of the stable positivity was extended to 7 to 8 days. In conclusion, the addition of a pyridine base such as pyridine or 4-methyl pyridine to the dithizone vital stain solution was effective not only in enhancing the positive staining but also in preventing the color from fading for 7 to 8 days due to the adduct formation. This concept for the stabilization of colored chelates may be applicable to other metal-staining methods. 1. Sumi, Y. (1993) Bunseki 11, 868–874. 2. Wolff, H., and Ringer, D. (1954) Naturwissenschaften 41, 260– 261. 3. Wolff, H., and Ringleb, D. (1954) Z. Ges. Exp. Med. 124, 236– 256. 4. Yoshinaga, T., and Yoshitake, S. (1963) Acta Histochem. 16, 337– 342. 5. Yoshinaga, T., Katayama, T., and Yamamoto, Y. (1965) Acta Histochem. 21, 276–283. 6. Akaiwa, H. (1972) Chushutu Bunri Bunsekiho (in Japanese), Kodansha, Tokyo. 7. Akaiwa, H., and Kawamoto, H. (1982) Rev. Anal. Chem. 6, 65– 86. 8. Akaiwa, H., Kawamoto, H., and Suzuki, K. (1983) Bunseki Kagaku 32, E109–E114.
Catalytic Hydrogenation of Leukotriene B4 Enhances Sensitivity and Specificity of Gas Chromatography–Tandem Mass Spectrometry Techniques and Enables Simultaneous Analysis with Cysteinyl Leukotrienes in Biological Fluids Dimitrios Tsikas,1 Joachim Fauler, Rube´n D. Vela´squez, Frank-Mathias Gutzki, and Ju¨rgen C. Fro¨lich Institute of Clinical Pharmacology, Hannover Medical School, Konstanty-Gutschow-Strasse 8, D-30625 Hannover, Germany Received September 7, 1995
Leukotriene (LT)2 B4 and the cysteinyl leukotrienes C 4 , D4 , and E 4 are potent mediators of in1
To whom correspondence should be addressed. Fax: 49/511/532– 2750. 2 Abbreviations used: LT, leukotriene; NICI, negative-ion chemical ionization; PFB, pentafluorobenzyl; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CAD, collisionally activated dissociation; SRM, ARTICLE NO.
flammation and anaphylaxis (1). Their analysis in biological fluids provides a method for assessing in vivo leukotriene production in humans (2). Gas chromatography – mass spectrometry (GC – MS) and GC –tandem MS techniques in the negative-ion chemical ionization (NICI) mode have been shown to allow quantification of leukotrienes in human serum, urine, and synovial fluid (3 – 10). The present study demonstrates that catalytic hydrogenation, which is indispensable for GC – MS analysis of cysteinyl leukotrienes (4) but not of LTB4 , both enhances sensitivity and specificity of the GC – tandem MS technique for LTB4 and allows its simultaneous analysis with cysteinyl leukotrienes in biological fluids. Materials and Methods
REFERENCES
ANALYTICAL BIOCHEMISTRY
175
Materials. LTB4 , LTE4 , and [1,2-13C2]LTB4 (99.8% at 13C by GC–MS) were obtained from Sigma Chemie (Munich, Germany). [20,20,20-2H3]LTE4 (99.8% at 2H by GC–MS) was purchased from Biomol (Hamburg, Germany). [5,6,8,9,11,12,14,15-3H8]LTB4 (100 Ci/ mmol) was obtained from Du Pont de Nemours (Dreieich, Germany). Pentafluorobenzyl (PFB) bromide and 12-hydroxyoctadecanoic acid were bought from Aldrich (Steinheim, Germany). N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) was obtained from Pierce (Rockford, IL). The catalyst used for catalytic hydrogenation (5 wt% Rh on Al2O3) was supplied by Fluka (Neu-Ulm, Germany). Gas chromatography–mass spectrometry and gas chromatography – tandem mass spectrometry. GC– MS was carried out on a Hewlett – Packard MS Engine 5989A directly connected with a gas chromatograph 5890 series II (Waldbronn, Germany). The gas chromatograph was equipped with a fused-silica capillary column DB-1 (15 m 1 0.25 mm i.d., 0.25-mm film thickness) from J&W Scientific (Rancho Cordova, CA) which was held at 1007C. Helium (35 kPa) and methane (200 Pa) were used as carrier and reagent gases, respectively. Interface, injector, and ion source were kept at 280, 250, and 2257C, respectively. Electron energy and electron current were set to 230 eV and 300 mA, respectively. The following oven temperature program was used: 2 min at 1007C, then increase to 2507C at a rate of 257C/min, followed by an increase to 3207C at a rate of 47C/min. GC – tandem MS was performed on a triple-stage quadrupole mass spectrometer Finnigan MAT TSQ 45 interfaced with a Finnigan MAT gas chromatograph 9611 (San Jose, CA). The same fused-silica capillary column and the same oven temperature program were selected reaction monitoring; SIM, selected ion monitoring; TMS, trimethylsilyl.
236, 175–178 (1996)
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m13$$$nat
03-13-96 14:21:48
abnt
AP-Anal Bio
was able to detect some other HA-binding proteins of high molecular weights also (Fig. 4C). It is also important to mention that the signal obtained by radioiodinated adduct is much more prominent compared to immunodetection, suggesting the high sensitivity of this method. The clinical importance of HA and its binding proteins is gaining importance due to its involvement in various diseases (1). The present method has advantages over the immunodetection method because it can detect a wide range of HA-binding proteins. In this regard, the applicability of this radioactive probe increases substantially and, consequently, it may have much wider applications in the future. REFERENCES 1. Knudson, C. B., and Knudson, W. (1993) FASEB J. 7, 1233– 1241. 2. Raja, R. H., Leboeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168–177. 3. Cardin, A. D., Witt, K. R., and Jackson, R. L. (1984) Anal. Biochem. 137, 368–373. 4. Yang, B., Hall, C. L., Yang, B. L., Savani, R. C., and Turley, E. A. (1994) J. Cell. Biochem. 56, 455–468. 5. Yu, Q., and Toole, B. P. (1995) Biotechniques 19, 122–129. 6. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849–857. 7. Bitter, T., and Muir, H. M. (1962) Anal. Biochem. 4, 330–334. 8. Laemmli, U. K. (1970) Nature 227, 680–685. 9. Gupta, S., Babu, B. R., and Datta, K. (1991) Eur. J. Cell. Biol. 56, 58–67. 10. Tengblad, A. (1979) Biochim. Biophys. Acta 578, 281–289. 11. Deb, T. B., and Datta, K. (1995) J. Biol. Chem., in press.
Enhancement and Stabilization of Dithizone Vital Staining for Zinc in Rat Organs Using Adduct Formation Chikako Shima, Kin-ichi Tsunoda, Hideo Akaiwa,1 Keiji Suzuki,* and Katsuyuki Nakajima† Department of Chemistry, Gunma University, Kiryu 376, Japan; *Department of Pathology, Medical Care and Technology, Gunma University, Maebashi 371, Japan; and †Japan Immunoresearch Laboratories Co., Ltd., Takasaki 370, Japan Received September 5, 1995
Zinc is one of the essential elements for mammalian species including human; numerous zinc enzymes have 1
To whom correspondence should be addressed. Fax: 81-277-301251. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
been found during the past several decades. Thus, the distribution of zinc in mammalian bodies has attracted wide attention in biomedical fields to elucidate its physiological role and metabolism. Histochemical staining techniques have often been used for such purposes (1). For example, the existence of zinc in the islet of Langerhans was shown by a dithizone staining method for zinc(II) (2), which has still been one of the most important staining methods for zinc in spite of some shortcomings such as its relatively low sensitivity (1–5). We have also applied the dithizone method to elucidate the zinc distribution in rat tissues. However, the ordinary staining method was not sensitive enough to detect zinc in a normal rat, even in tissues such as pancreas and prostate which are known to concentrate zinc (1). Thus, the vital staining method was applied to this problem (2– 5). Although violet color development due to the formation of zinc(II)–dithizonate was observed in several organs by this method, the positive staining faded out almost completely half a day after the specimen preparation because of the decomposition of the complex. In this paper, we overcome this problem by establishing an improved dithizone vital staining method in which adduct formation of zinc(II)–dithizonate with pyridine or 4-methyl pyridine was applied to stabilize the complex. With the present method, the positive staining was strongly enhanced and the period of the stable positivity was extended up to 7 to 8 days. All chemical reagents used in this study were analytical grade and used without further purification. Dithizone stain solution A. Dithizone (100 mg) was dissolved in 1 ml ethanol with 0.2 ml concentrated ammonia solution. Then the solution was diluted with 20 ml deionized water. Dithizone stain solution B. Pyridine base, i.e., pyridine or 4-methyl pyridine whose concentration was equivalent to or five times as high as dithizone, was added to the solution A. Dithizone vital staining of rat tissues for zinc (4–6). Male rats weighing ca. 200 g were used for all experiments. The dithizone stain solution A or B (3–4 ml) was infused by intraperitoneal injection into a rat anesthetized with ether. After 30 min, the organs were taken out, frozen with liquid nitrogen, then cut at a thickness of 3 mm with a cryostat. The sliced tissues were stained with methyl green and enclosed with a glycerol and gelatin mixture on a glass slide. The examined organs were brain, salivary gland, lung, thymus, heart, spleen, liver, pancreas, kidney, intestine, testis, and prostate. Among them, the violet color development due to zinc(II)–dithizonate complex was found in the pancreas (the islet of Langerhans), prostate (epithelial cells of lateral lobe), and intestine (Paneth’s cells) with the ordinary dithizone vital staining method (2–5). Figure 1 is a photomicrograph of prostate as an example. Because
236, 173–175 (1996)
0148
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m13$$$nat
03-13-96 14:21:48
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173
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174
NOTES & TIPS
FIG. 1. Photomicrograph of rat prostate with dithizone vital staining (immediately after specimen preparation). No pyridine base was added to the stain solution. Epithelial cells are positive. Original magnification 1100.
of the decomposition of the complex, however, the violet color faded out almost completely half a day after the specimen preparation (Fig. 2). Thus, we tried to stabilize the Zn(II)–dithizonate and to prolong the positivity by the adduct formation with pyridine bases; the residual water molecules which coordinate to the central metal ion in the complex are replaced by the pyridine base, thus resulting in higher stability of the complex due to the prevention of oxidation reaction as well as the increase in its hydrophobicity (6). In this study, pyridine and 4-methyl pyridine were chosen as the pyridine base, because they have often been used as an adduct formation agent in solvent extraction of metal ions (7, 8). In general, 4-methyl pyridine is a better adduct-forming agent than pyridine because of its stronger basicity (6). At first, the sliced vital stained tissue was dipped in these pyridine base solutions. However, no
FIG. 2. Photomicrograph of rat prostate with dithizone vital staining (3 days after specimen preparation, the same specimen as that of Fig. 1). Positive staining of epithelial cells faded out gradually. Original magnification 1100.
/ 6m13$$$nat
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FIG. 3. Photomicrograph of rat prostate with dithizone vital staining (immediately after specimen preparation). 4-Methyl pyridine was added to the stain solution at the concentration equivalent to that of dithizone. Epithelial cells are strongly positive. Original magnification 1400.
effect was observed on the prevention of the positive staining from fading in this in vitro experiment. Thus, pyridine bases were added to the dithizone stain solution and injected intraperitoneally into the rat together with dithizone. The concentration of each base was the same or five times as high as that of dithizone. A remarkable enhancement of and stabilizing effect on the positive staining was observed in every case. The effects were more remarkable with 4-methyl pyridine than with pyridine as expected from their adduct-forming abilities. With the addition of the equivalent concentration of 4-methyl pyridine to dithizone, the stable positivity of zinc(II)–dithizonate lasted for up to 6 days. Figures 3 and 4 are photomicrographs of the vital stained rat prostate with dithizone and 4-methyl pyridine (equivalent concentration to dithizone), immediately after specimen preparation and after 6 days, respec-
FIG. 4. Photomicrograph of rat prostate with dithizone vital staining (6 days after specimen preparation, the same specimen as Fig. 4). Original magnification 1400.
AP-Anal Bio
NOTES & TIPS
tively. The dark violet granules can be observed in these figures. Essentially, the degree of the positive staining was much greater in Figs. 3 and 4 than in Fig. 1. The effect of 4-methyl pyridine on the prevention of the color from fading (Fig. 4) was also remarkable compared with Fig. 2. Moreover, with the addition of a five times higher concentration of 4-methyl pyridine, the positivity was further strongly enhanced and the period of the stable positivity was extended to 7 to 8 days. In conclusion, the addition of a pyridine base such as pyridine or 4-methyl pyridine to the dithizone vital stain solution was effective not only in enhancing the positive staining but also in preventing the color from fading for 7 to 8 days due to the adduct formation. This concept for the stabilization of colored chelates may be applicable to other metal-staining methods. 1. Sumi, Y. (1993) Bunseki 11, 868–874. 2. Wolff, H., and Ringer, D. (1954) Naturwissenschaften 41, 260– 261. 3. Wolff, H., and Ringleb, D. (1954) Z. Ges. Exp. Med. 124, 236– 256. 4. Yoshinaga, T., and Yoshitake, S. (1963) Acta Histochem. 16, 337– 342. 5. Yoshinaga, T., Katayama, T., and Yamamoto, Y. (1965) Acta Histochem. 21, 276–283. 6. Akaiwa, H. (1972) Chushutu Bunri Bunsekiho (in Japanese), Kodansha, Tokyo. 7. Akaiwa, H., and Kawamoto, H. (1982) Rev. Anal. Chem. 6, 65– 86. 8. Akaiwa, H., Kawamoto, H., and Suzuki, K. (1983) Bunseki Kagaku 32, E109–E114.
Catalytic Hydrogenation of Leukotriene B4 Enhances Sensitivity and Specificity of Gas Chromatography–Tandem Mass Spectrometry Techniques and Enables Simultaneous Analysis with Cysteinyl Leukotrienes in Biological Fluids Dimitrios Tsikas,1 Joachim Fauler, Rube´n D. Vela´squez, Frank-Mathias Gutzki, and Ju¨rgen C. Fro¨lich Institute of Clinical Pharmacology, Hannover Medical School, Konstanty-Gutschow-Strasse 8, D-30625 Hannover, Germany Received September 7, 1995
Leukotriene (LT)2 B4 and the cysteinyl leukotrienes C 4 , D4 , and E 4 are potent mediators of in1
To whom correspondence should be addressed. Fax: 49/511/532– 2750. 2 Abbreviations used: LT, leukotriene; NICI, negative-ion chemical ionization; PFB, pentafluorobenzyl; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CAD, collisionally activated dissociation; SRM, ARTICLE NO.
flammation and anaphylaxis (1). Their analysis in biological fluids provides a method for assessing in vivo leukotriene production in humans (2). Gas chromatography – mass spectrometry (GC – MS) and GC –tandem MS techniques in the negative-ion chemical ionization (NICI) mode have been shown to allow quantification of leukotrienes in human serum, urine, and synovial fluid (3 – 10). The present study demonstrates that catalytic hydrogenation, which is indispensable for GC – MS analysis of cysteinyl leukotrienes (4) but not of LTB4 , both enhances sensitivity and specificity of the GC – tandem MS technique for LTB4 and allows its simultaneous analysis with cysteinyl leukotrienes in biological fluids. Materials and Methods
REFERENCES
ANALYTICAL BIOCHEMISTRY
175
Materials. LTB4 , LTE4 , and [1,2-13C2]LTB4 (99.8% at 13C by GC–MS) were obtained from Sigma Chemie (Munich, Germany). [20,20,20-2H3]LTE4 (99.8% at 2H by GC–MS) was purchased from Biomol (Hamburg, Germany). [5,6,8,9,11,12,14,15-3H8]LTB4 (100 Ci/ mmol) was obtained from Du Pont de Nemours (Dreieich, Germany). Pentafluorobenzyl (PFB) bromide and 12-hydroxyoctadecanoic acid were bought from Aldrich (Steinheim, Germany). N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) was obtained from Pierce (Rockford, IL). The catalyst used for catalytic hydrogenation (5 wt% Rh on Al2O3) was supplied by Fluka (Neu-Ulm, Germany). Gas chromatography–mass spectrometry and gas chromatography – tandem mass spectrometry. GC– MS was carried out on a Hewlett – Packard MS Engine 5989A directly connected with a gas chromatograph 5890 series II (Waldbronn, Germany). The gas chromatograph was equipped with a fused-silica capillary column DB-1 (15 m 1 0.25 mm i.d., 0.25-mm film thickness) from J&W Scientific (Rancho Cordova, CA) which was held at 1007C. Helium (35 kPa) and methane (200 Pa) were used as carrier and reagent gases, respectively. Interface, injector, and ion source were kept at 280, 250, and 2257C, respectively. Electron energy and electron current were set to 230 eV and 300 mA, respectively. The following oven temperature program was used: 2 min at 1007C, then increase to 2507C at a rate of 257C/min, followed by an increase to 3207C at a rate of 47C/min. GC – tandem MS was performed on a triple-stage quadrupole mass spectrometer Finnigan MAT TSQ 45 interfaced with a Finnigan MAT gas chromatograph 9611 (San Jose, CA). The same fused-silica capillary column and the same oven temperature program were selected reaction monitoring; SIM, selected ion monitoring; TMS, trimethylsilyl.
236, 175–178 (1996)
0149
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m13$$$nat
03-13-96 14:21:48
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