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A Routine Assay for Methoxytryptophol and Melatonin in the Peripheral Circulation using Gas Chromatography-Mass Spectrometry
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A Routine Assay for Methoxytryptophol and Melatonin in the Peripheral Circulation using Gas Chromatography-Mass Spectrometry R.M. LEONE, R.E. SILMAN, R.J.L. HOOPER, S.J. CARTER,M.D.A. FINNIE, R. EDWARDS, I. SMITH l , P. FRANCIS and P.E. MULLEN Departments of Chemical Pathology and Reproductive Physiology, St. Bartholomew's Hospital Medical College, London EC I ; The Courtauld Institute, Middlesex Hospital Medical School, London W I ; and Institute of Psychiatry, Maudsley Hospital, De Crespigny Park, London SE 5 (Great Britain)
The role of the pineal gland in human physiology is unclear and this is due, in part, to difficulties in measuring its hormonal output in the peripheral circulation. Though bioassays and immunoassays now exist for melatonin (aMT), other methoxyindoles which are synthesized by the pineal gland have received little attention, Using gas chromatography-mass spectrometry (GCMS), we have developed an assay for 5-methoxytryptophol (ML) which has great sensitivity and unparalleled specificity (Leone et al., 1979). However, GCMS offers the further advantage of versatility, and the technology can be extended to include all pineal indoles. We present an account of its routine application for the combined assay of ML and aMT in single plasma samples. Fifty ng of synthetic internal standard [3(butan-3'-one) 5-methoxyindole] was added to 1 ml plasma. Samples were vortexed for 10 sec with 3 ml redistilled chloroform and spun at 3000 rpm for 10 min. The organic phase was removed and blown to dryness under nitrogen to be reconstituted in 5 pl pyridine and 15 /.d trimethylsilyl (TMS) reagent. Five /.d aliquots (equivalent to 0.25 ml plasma) were injected onto a Varian 1440 GC coupled to a Varian Mat 3 11A MS. The 2-m column was packed with 10%OVI. The oven temperature was set at 280°C for 3 min to allow the elution of ML and the internal standard, and then programmed to 350°C at lO"C/min for the elution of aMT. The eluate was monitored at mass 232, the characteristic fragment ion of the methoxyindole nucleus. The standard curve was plotted using a fixed amount of internal standard against varying amounts of ML or aMT, the ratio of their peak heights to that of the internal standard being proportional to the concentration of ML or aMT. Figure 1 shows a standard curve for ML. The assay has good precision since extraction losses and slight alterations of GCMS conditions are normalised by reference to the internal standard. The specificity of the assay is guaranteed by monitoring at mass 232. But further validation is possible. For example, aMT has a small molecular'ion of mass 304 and can be monitored instead at this mass number. Figure 2 illustrates the measurement of melatonin from plasma taken at different times during an insulin induced hypoglycaemic stress test. Though the levels of aMT vary at different moments in the test, measurement by both assays (mass 232 and mass 304) show similar results. Until recently, GCMS was considered a secondary technology, something that could be used to validate alternative assay systems. But its flexibility and the development of assays which are assured of great precision, sensitivity and specificity now make it the method of choice for the measurement of pineal indoles.
264
100 ratio of internal standard to ML
10
1
0.8 0.6 0.5 0.4
0.3 0.2 0.1
1oPg
loopa
1n9
CONCENTRATION OF MU5pl injection
Fig. 1. The standard curve for 5-methoxytryptophol (ML)was obtained by calculating the ratio of the peak heights of varying amounts of ML to a constant amount of internal standard. The figures under each point represent the numbers of observations and the bars represent the standard errors of the mean. (From Leone et al., 1979.)
"1
Fig. 2. Blood was taken at 6 different times during an insulin-induced hypoglycaemic stress test and melatonin assayed at mass 232 and at mass 304.
265
ACKNOWLEDGEMENT This work has been generously supported by an interdepartmental grant from the Wellcome Trust to study the endocrine role of the pineal gland in man. REFERENCE Leone, R.M., Silman, R.E., Hooper, R.J.L.,Finnie, M.D.A.,Carter, S.J., Edwards, R., Smith, I., Towell, P. and Mullen, P.E. (1979) A sensitive and specific assay for 5-methoxytryptophol in plasma. J. Endocrinol., In press.
A Routine Assay for Methoxytryptophol and Melatonin in the Peripheral Circulation using Gas Chromatography-Mass Spectrometry R.M. LEONE, R.E. SILMAN, R.J.L. HOOPER, S.J. CARTER,M.D.A. FINNIE, R. EDWARDS, I. SMITH l , P. FRANCIS and P.E. MULLEN Departments of Chemical Pathology and Reproductive Physiology, St. Bartholomew's Hospital Medical College, London EC I ; The Courtauld Institute, Middlesex Hospital Medical School, London W I ; and Institute of Psychiatry, Maudsley Hospital, De Crespigny Park, London SE 5 (Great Britain)
The role of the pineal gland in human physiology is unclear and this is due, in part, to difficulties in measuring its hormonal output in the peripheral circulation. Though bioassays and immunoassays now exist for melatonin (aMT), other methoxyindoles which are synthesized by the pineal gland have received little attention, Using gas chromatography-mass spectrometry (GCMS), we have developed an assay for 5-methoxytryptophol (ML) which has great sensitivity and unparalleled specificity (Leone et al., 1979). However, GCMS offers the further advantage of versatility, and the technology can be extended to include all pineal indoles. We present an account of its routine application for the combined assay of ML and aMT in single plasma samples. Fifty ng of synthetic internal standard [3(butan-3'-one) 5-methoxyindole] was added to 1 ml plasma. Samples were vortexed for 10 sec with 3 ml redistilled chloroform and spun at 3000 rpm for 10 min. The organic phase was removed and blown to dryness under nitrogen to be reconstituted in 5 pl pyridine and 15 /.d trimethylsilyl (TMS) reagent. Five /.d aliquots (equivalent to 0.25 ml plasma) were injected onto a Varian 1440 GC coupled to a Varian Mat 3 11A MS. The 2-m column was packed with 10%OVI. The oven temperature was set at 280°C for 3 min to allow the elution of ML and the internal standard, and then programmed to 350°C at lO"C/min for the elution of aMT. The eluate was monitored at mass 232, the characteristic fragment ion of the methoxyindole nucleus. The standard curve was plotted using a fixed amount of internal standard against varying amounts of ML or aMT, the ratio of their peak heights to that of the internal standard being proportional to the concentration of ML or aMT. Figure 1 shows a standard curve for ML. The assay has good precision since extraction losses and slight alterations of GCMS conditions are normalised by reference to the internal standard. The specificity of the assay is guaranteed by monitoring at mass 232. But further validation is possible. For example, aMT has a small molecular'ion of mass 304 and can be monitored instead at this mass number. Figure 2 illustrates the measurement of melatonin from plasma taken at different times during an insulin induced hypoglycaemic stress test. Though the levels of aMT vary at different moments in the test, measurement by both assays (mass 232 and mass 304) show similar results. Until recently, GCMS was considered a secondary technology, something that could be used to validate alternative assay systems. But its flexibility and the development of assays which are assured of great precision, sensitivity and specificity now make it the method of choice for the measurement of pineal indoles.
264
100 ratio of internal standard to ML
10
1
0.8 0.6 0.5 0.4
0.3 0.2 0.1
1oPg
loopa
1n9
CONCENTRATION OF MU5pl injection
Fig. 1. The standard curve for 5-methoxytryptophol (ML)was obtained by calculating the ratio of the peak heights of varying amounts of ML to a constant amount of internal standard. The figures under each point represent the numbers of observations and the bars represent the standard errors of the mean. (From Leone et al., 1979.)
"1
Fig. 2. Blood was taken at 6 different times during an insulin-induced hypoglycaemic stress test and melatonin assayed at mass 232 and at mass 304.
265
ACKNOWLEDGEMENT This work has been generously supported by an interdepartmental grant from the Wellcome Trust to study the endocrine role of the pineal gland in man. REFERENCE Leone, R.M., Silman, R.E., Hooper, R.J.L.,Finnie, M.D.A.,Carter, S.J., Edwards, R., Smith, I., Towell, P. and Mullen, P.E. (1979) A sensitive and specific assay for 5-methoxytryptophol in plasma. J. Endocrinol., In press.