The Analysis of Thiocarbamides by - Gas Chromatography /Negative-ion Chemical-ionization Mass Spectrometry D. G. Watson,* C. D. Bates and G. G . Skellern Biomedical GUMS Unit, Department of Pharmacy, University of Strathclyde, Glasgow G1 IXW, UK
R. Mairs and S. Martin Department of Radiation Oncology, CRC Beatson Laboratories, Garscube Estate, Glasgow G61 lBD, UK SPONSOR REFEREE: Professor C. J. W. Brooks, Chemistry Department, University of Glasgow, Glasgow, UK
Thiocarbamides were converted to their di-N-pentafluorobenzyl (PFB) derivatives and analysed by gas chromatographyhegative-ion chemical-ionization mass spectrometry with methane as reagent gas. The PFB derivatives of the 2-thiouracils gave mass spectra in which the ion current was carried largely by an ion arising from [M - PFBI-. The derivative was used in the determinationof the uptake and metabolism of thiocarbamides by cultures of melanoma cells.
Evidence for the selective incorporation of thiocarbamides into abnormal melanosomes in melanoma cultures, has been based on the uptake of radioactivity when the cultures are exposed to radio-labelled thiocarbamides. ', These compounds have the potential to interfere with melanin biosynthesis through the formation of adducts with the reactive quinone intermediates involved in the biosynthesis. This may make them useful as therapeutic or drug-targeting agents in the treatment of melanoma. We have therefore developed a specific and sensitive GUMS procedure for studying the uptake of a range of substituted 2thiouracils by melanoma cells. EXPERIMENTAL Gas chromatographyhegative-ion chemical-ionization (GC/NICI) mass spectrometry was carried out using a Type 5988A gas chromatography/mass spectrometry system (Hewlett Packard Co., Palo Alto, CA, USA). Methane was used as a reagent gas with a source pressure ca 2.0Torr and a source temperature of 140°C. The gas chromatograph was fitted with an AQ21BP1 column (length 12 m x 0.25 mm ID) (SGE, Milton Keynes, UK) and the temperature was programmed from 100"C at 20 "C min-'. di-N-pentafluorobenzyl (PFB) derivatives of the 2thiouracils were prepared as follows: the thiouracil (50 pg) was dissolved in acetonitrile (30 pL). Triethylamine (5 pL) and pentafluorobenzyl bromide (5 pL) were added. The mixture was allowed to stand for 5 min at room temperature and was then diluted with ethyl acetate (200 pL) followed by hexane (600pL). The mixture was left to stand for ca 2min while a white precipitate formed. It was then filtered through a short column of Sephadex LH20 (2 cm in a Pasteur pipette) and the column was washed with hexane (1 mL). The solution was evaporated under a stream of nitrogen to 0.5 mL and 1pL of the final solution was injected into the G U M S system. In order to quantify 2-thiouracil in tissues and incubation Author to whom correspondence should be addressed.
[M -PF$
307
100
200
150
250
300
350
m/z
Figure 1. NICIMS of thiouracil diPFB derivative.
medium a calibration curve was constructed by derivatizing a fixed amount of methylthiouracil (4 pg) with varying amounts (200 ng-2 pg) of thiouracil or propylthiouracil. The [M-PFBI- ions were monitored for both of the thiouracils and comparison of the area ratios yielded almost linear calibration curves: thiouracil (correlation coefficient = 0.990), propylthiouracil (correlation coefficient = 0.987). RESULTS AND DISCUSSION The NICI mass spectra of the di-N-PFB derivatives of 2-thiouracil and 6-methyl-2-thiouracil are shown in Figs. 1 and 2. The ion current, in the case of methylthiouracil, is carried almost entirely by an ion arising [M --pFB]'
100
150
200
250
1 300
350
400
m/z
Figure 2. NICIMS of benzylthiouracil diPFB derivative.
0951-4198/91/0141-0142$05.00
0
John Wiley & Sons Limited, 1991
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5. NO. 3, 1991 141
CI SPECTRA OF PENTAFLUOROBENZYL DERIVATIVES OF THIOCARBAMIDES 4TU
Table 1. GClNICIMS data for thiocarbamidesas PFB derivatives Compound
Methimazole 2-Thiouracil 6-Methylthiouracil 5-Propylthiouracil 6-Propylthiouracil 6-Benzylthiouracil
Retention index
Base peak m / z
1625 2305 2343 2444 2450 2782
113 307 321 349 349 396
Other ions >5%
127 (17.5) 141 (6) 169 (15) 169 (14) 217 (10)
from the loss of PFB from M-’; in the case of thiouracil some of the ion current is carried by a reagent-specific ion at mlz181 where the PFE3 moiety carries the charge. This type of spectrum was observed previously where di-N-PEB derivatives of 5-flU01-0- and 5chlorouracil were prepared and analysed under NICI condition^.^ Table 1 summarizes retention index data and the mass spectra for four 2-thiouracils and for 1methyl-2-thioimidazole (methimazole). (PFB)Br has been used previously to alkylate methimazole prior to analysis by GC; it was thought that the derivative in this Figure 3 shows case was S- rather than N-alk~lated.~ the gas chromatogram of propylthiouracil (PTU), as its PFB derivative, extracted from melanoma cells, after they had been incubated with propylthiouracil (40pgmL-I in the culture medium) for 24 h, in comparison with 6-methylthiouracil (MTU) (4 pg) added to the cells before extraction. Uptake of thiouracil by live cells was only slightly different from that by dead cells. However, the uptake appears to be related to lipophilicity since 6-propylthiouracil was taken up by live melanoma cells to a significantly greater extent than by the dead. These results will be reported in full elsewhere. To achieve good chromatographic peak shape with this derivative it was necessary to run samples on a clean column reserved specifically for the analysis. The peak shape of the derivative may be adversely affected by polar materials adsorbed onto the column from previous runs. The high sensitivity afforded by the
10.0
11.0
12.0
min
Figure 3. SIM trace monitoring the ions for methylthiouracil (mlz 321) and propylthiouracil ( m / z 349).
derivative (theoretical limit of detection ca 1pg, practical limit of detection ca 100 pg) enabled extracts from biological samples in dilute solution to be injected onto the column thus extending its lifetime. Acknowledgements We thank the Scottish Home and Health Department for financial support.
REFERENCES 1. R. A. Farishian and J. R. Whittaker, Arch. Biochem. Biophys. 198, 449 (1989). 2. P. G. Parsons and B. J. Allen, Austral. J. Exp. Biol. Med. Sci. 64, 517 (1986). 3. R. M. Kok, A. P. J. M. De Jong, C. J. Van Groeningen, G. J. Peters and J. Lankelma, J. Chromatogr. Biomed. Appl. 221, 382 (1985). 4. S. Floberg, K. Lanbeck and B. Lindstrom. J. Chromatogr. 182,63 (1980). Received 25 January 1991; accepted 27 January 1991.
142 RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5, NO. 3, 1991
@ John Wiley & Sons Limited, 1991
R. Mairs and S. Martin Department of Radiation Oncology, CRC Beatson Laboratories, Garscube Estate, Glasgow G61 lBD, UK SPONSOR REFEREE: Professor C. J. W. Brooks, Chemistry Department, University of Glasgow, Glasgow, UK
Thiocarbamides were converted to their di-N-pentafluorobenzyl (PFB) derivatives and analysed by gas chromatographyhegative-ion chemical-ionization mass spectrometry with methane as reagent gas. The PFB derivatives of the 2-thiouracils gave mass spectra in which the ion current was carried largely by an ion arising from [M - PFBI-. The derivative was used in the determinationof the uptake and metabolism of thiocarbamides by cultures of melanoma cells.
Evidence for the selective incorporation of thiocarbamides into abnormal melanosomes in melanoma cultures, has been based on the uptake of radioactivity when the cultures are exposed to radio-labelled thiocarbamides. ', These compounds have the potential to interfere with melanin biosynthesis through the formation of adducts with the reactive quinone intermediates involved in the biosynthesis. This may make them useful as therapeutic or drug-targeting agents in the treatment of melanoma. We have therefore developed a specific and sensitive GUMS procedure for studying the uptake of a range of substituted 2thiouracils by melanoma cells. EXPERIMENTAL Gas chromatographyhegative-ion chemical-ionization (GC/NICI) mass spectrometry was carried out using a Type 5988A gas chromatography/mass spectrometry system (Hewlett Packard Co., Palo Alto, CA, USA). Methane was used as a reagent gas with a source pressure ca 2.0Torr and a source temperature of 140°C. The gas chromatograph was fitted with an AQ21BP1 column (length 12 m x 0.25 mm ID) (SGE, Milton Keynes, UK) and the temperature was programmed from 100"C at 20 "C min-'. di-N-pentafluorobenzyl (PFB) derivatives of the 2thiouracils were prepared as follows: the thiouracil (50 pg) was dissolved in acetonitrile (30 pL). Triethylamine (5 pL) and pentafluorobenzyl bromide (5 pL) were added. The mixture was allowed to stand for 5 min at room temperature and was then diluted with ethyl acetate (200 pL) followed by hexane (600pL). The mixture was left to stand for ca 2min while a white precipitate formed. It was then filtered through a short column of Sephadex LH20 (2 cm in a Pasteur pipette) and the column was washed with hexane (1 mL). The solution was evaporated under a stream of nitrogen to 0.5 mL and 1pL of the final solution was injected into the G U M S system. In order to quantify 2-thiouracil in tissues and incubation Author to whom correspondence should be addressed.
[M -PF$
307
100
200
150
250
300
350
m/z
Figure 1. NICIMS of thiouracil diPFB derivative.
medium a calibration curve was constructed by derivatizing a fixed amount of methylthiouracil (4 pg) with varying amounts (200 ng-2 pg) of thiouracil or propylthiouracil. The [M-PFBI- ions were monitored for both of the thiouracils and comparison of the area ratios yielded almost linear calibration curves: thiouracil (correlation coefficient = 0.990), propylthiouracil (correlation coefficient = 0.987). RESULTS AND DISCUSSION The NICI mass spectra of the di-N-PFB derivatives of 2-thiouracil and 6-methyl-2-thiouracil are shown in Figs. 1 and 2. The ion current, in the case of methylthiouracil, is carried almost entirely by an ion arising [M --pFB]'
100
150
200
250
1 300
350
400
m/z
Figure 2. NICIMS of benzylthiouracil diPFB derivative.
0951-4198/91/0141-0142$05.00
0
John Wiley & Sons Limited, 1991
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5. NO. 3, 1991 141
CI SPECTRA OF PENTAFLUOROBENZYL DERIVATIVES OF THIOCARBAMIDES 4TU
Table 1. GClNICIMS data for thiocarbamidesas PFB derivatives Compound
Methimazole 2-Thiouracil 6-Methylthiouracil 5-Propylthiouracil 6-Propylthiouracil 6-Benzylthiouracil
Retention index
Base peak m / z
1625 2305 2343 2444 2450 2782
113 307 321 349 349 396
Other ions >5%
127 (17.5) 141 (6) 169 (15) 169 (14) 217 (10)
from the loss of PFB from M-’; in the case of thiouracil some of the ion current is carried by a reagent-specific ion at mlz181 where the PFE3 moiety carries the charge. This type of spectrum was observed previously where di-N-PEB derivatives of 5-flU01-0- and 5chlorouracil were prepared and analysed under NICI condition^.^ Table 1 summarizes retention index data and the mass spectra for four 2-thiouracils and for 1methyl-2-thioimidazole (methimazole). (PFB)Br has been used previously to alkylate methimazole prior to analysis by GC; it was thought that the derivative in this Figure 3 shows case was S- rather than N-alk~lated.~ the gas chromatogram of propylthiouracil (PTU), as its PFB derivative, extracted from melanoma cells, after they had been incubated with propylthiouracil (40pgmL-I in the culture medium) for 24 h, in comparison with 6-methylthiouracil (MTU) (4 pg) added to the cells before extraction. Uptake of thiouracil by live cells was only slightly different from that by dead cells. However, the uptake appears to be related to lipophilicity since 6-propylthiouracil was taken up by live melanoma cells to a significantly greater extent than by the dead. These results will be reported in full elsewhere. To achieve good chromatographic peak shape with this derivative it was necessary to run samples on a clean column reserved specifically for the analysis. The peak shape of the derivative may be adversely affected by polar materials adsorbed onto the column from previous runs. The high sensitivity afforded by the
10.0
11.0
12.0
min
Figure 3. SIM trace monitoring the ions for methylthiouracil (mlz 321) and propylthiouracil ( m / z 349).
derivative (theoretical limit of detection ca 1pg, practical limit of detection ca 100 pg) enabled extracts from biological samples in dilute solution to be injected onto the column thus extending its lifetime. Acknowledgements We thank the Scottish Home and Health Department for financial support.
REFERENCES 1. R. A. Farishian and J. R. Whittaker, Arch. Biochem. Biophys. 198, 449 (1989). 2. P. G. Parsons and B. J. Allen, Austral. J. Exp. Biol. Med. Sci. 64, 517 (1986). 3. R. M. Kok, A. P. J. M. De Jong, C. J. Van Groeningen, G. J. Peters and J. Lankelma, J. Chromatogr. Biomed. Appl. 221, 382 (1985). 4. S. Floberg, K. Lanbeck and B. Lindstrom. J. Chromatogr. 182,63 (1980). Received 25 January 1991; accepted 27 January 1991.
142 RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5, NO. 3, 1991
@ John Wiley & Sons Limited, 1991
