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Some observations on the t-butyldimethylchlorosilane derivatization reaction
BIOCHEMICAL
MEDICINE
23,
119-121
(1980)
SHORT COMMUNICATIONS Some Observations on the t-Butyldimethylchlorosilane Derivatization Reaction Recent studies conducted in our laboratory have involved the assay of the major urinary metabolite of PGF@ (PGF-M) as its tBDMS ether, using gas-liquid chromatography-mass spectrometry (glc-MS). Erratic results prompted investigation of the tBDMS reaction. We followed the urinary extraction and purification method of Brash e? al., l with some minor modifications, and after having several assays with a very low or zero recovery even of internal standard, decided to replace the urine with distilled water in order to reexamine the procedure step by step. Several aliquots of distilled water were “spiked” with a large amount (400 ng) of a d3 PGF-M methyl ester and processed according to the Brash technique. Samples were terminated at various stages of the method and derivatized for assay by glc-MS. From these experiments we were able to determine that the problem was associated with the final derivatization with rBDMS. EXPERIMENTAL Reagenrs. The tBDMS was obtained from two different suppliers, (1) a formulated tBDMS-imidazole in sealed ampoules from Applied Science Laboratories, and (2) crystalline tBDMS in pure form from Regis. Imidazole was from Matheson Coleman-Bell, dimethylformamide and 1-hexadecanol was from Eastman Organic Chemicals. The dimethylformamide was redistilled and dried over molecular sieve before using. Gus-liquid chromatography. A Varian model 2100 instrument was used for the initial screening runs, in which a rBDMS-hexadecanol reaction product was used to provide an immediate check on the formation of a tBDMS ether by each batch of reagent. A glass column 6 ft x 4mm i.d. was packed with 3% OV-1 on Chromosorb W AW 100-120 mesh and connected to the flame ionization detector (FID). Operating parameters were, nitrogen carrier gas 40 ml/mitt, injection port, oven, and detector temperatures at 230, 180, and 240°C respectively. FID range set at 10-I1 attenuation at 128X. 119 00062944/80/010119-03$02.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
120
SHORTCOMMUNICATIONS
A fresh batch of tBDMS-imidazole ampoules was obtained from Applied Science Laboratories, and the reagent transferred to a small Teflon-lined screw-cap vial under nitrogen and is referred to hereafter as Reagent A. Laboratory prepared solutions of the Regis tBDMS and the imidazole were made up in dry dimethylformamide (DMF) to the same final molar concentration as given by Applied Science with their ampoules, i.e., 1.0 mmole rBDMSlmilliliter and 2.5 mmole imidazolei milliliter. These solutions were used either as separate solutions to be mixed at the time of use (Reagent B) or as a premixed solution (Reagent C). All reagents were stored under nitrogen at - 12°C. 1-Hexadecanol was dissolved in dry DMF at a concentration of 0.1 mmole/milliliter and used to compare the reaction potency of the tBDMS reagent from both sources. A 20-~1 aliquot of hexadecanol solution was mixed with 20 ~1 of the reagent, or in the case of separate solutions, 10 ~1 each of tBDMS and imidazole. The reaction was done in small l-ml Teflon-lined screw-cap vials. The glc profiles were developed using l-w1 injections at zero reaction time and repeated at 24-hr reaction time at room temperature. Subsequent reaction mixtures were set up at various daily intervals to check the stability of the reagents. Having established optimum reaction conditions, urine samples containing the d3 PGF-M methyl ester “spike” were subjected to the extraction, purification, and derivatization procedure and assayed by glc-MS. RESULTS
Comparison of Reagent A versus Reagent B is shown in Fig. 1. Regis Lab formulated reagent produced a larger reaction product peak of tBDMS-hexadecanol derivative than did the Applied Science reagent. The locally formulated premixed Reagent C gave comparable results to Reagent B and “free” hexadecanol was absent with all three reagent reactions. Reassay of the same reaction mixtures at 24 hr, showed a diminished reaction product for both reagents A and B, with a corresponding increase of a secondary reaction peak at 1.5 min retention time. Stability of reagents A and B was good for several days, however, some unreacted hexadecanol was present using ‘I-day-old reagents. Reagent C (locally premixed) appeared to deteriorate much faster having no reaction potency at 7 days. The secondary reaction product peak at 1.5 min retention time was evident with all three reagents. It gradually increased in concentration in subsequent runs, as the reagents aged. The source of this peak appears to be a product of the tBDMS-imidazole mixture, since glc profiles of the two separate solutions even when 7 days old, showed no such peak. However, when mixed together and injected immediately, a peak did
121
SHORT COMMUNICATIONS
Id
FIG. 1. Gas-liquid chromatography traces of reaction product tBDMS-hexadecanol (retention time 11 mitt). (la) Reagent A (Applied Science) and (lb) Reagent B (Regis), immediate assay: (1~) Reagent A and (Id) Reagent B. repeat assay at 24 hr.
appear at the 1.5min retention time. No attempt was made to identify this secondary reaction product by glc-MS. Assay of the product of the “immediate reaction” using the spiked urine samples, instead of after an overnight reaction as originally described by Brash et al., yielded a 45-50% increase in reaction product as assayed with glc-MS. CONCLUSION
In conclusion, freshly prepared rBDMS-imidazole reagent is the most desirable one to use in terms of potency. Assay of *‘immediate reaction” samples result in higher yields of product from both hexadecanol and the prostaglandin F,a! metabolite. The Applied Science reagent in sealed ampoules give no formulation or expiration date and the lower yield may be due to aged reagents. ACKNOWLEDGMENTS We wish to express our thanks to Mr. R. W. Silverman for providing the glc-MS assays. Supported in part by USPHS-RR5354, American Heart Association LAS93, and a grant from Pharmaceutical Manufacturers Association Foundation.
REFERENCE 1. Brash, A. R., Baillie, T. A., Clam, R. A., and Draffan, G. A.,Eiochem. Med. 16,n (1976). KENNETH BRICKNELL MATTHEW E. CONOLLY
University of California at Los Angeles, School of Medicine, Division of Clinical Pharmacology, Department of Pharmacology, Los Angeles, California 90024 Received February 8, 1919
MEDICINE
23,
119-121
(1980)
SHORT COMMUNICATIONS Some Observations on the t-Butyldimethylchlorosilane Derivatization Reaction Recent studies conducted in our laboratory have involved the assay of the major urinary metabolite of PGF@ (PGF-M) as its tBDMS ether, using gas-liquid chromatography-mass spectrometry (glc-MS). Erratic results prompted investigation of the tBDMS reaction. We followed the urinary extraction and purification method of Brash e? al., l with some minor modifications, and after having several assays with a very low or zero recovery even of internal standard, decided to replace the urine with distilled water in order to reexamine the procedure step by step. Several aliquots of distilled water were “spiked” with a large amount (400 ng) of a d3 PGF-M methyl ester and processed according to the Brash technique. Samples were terminated at various stages of the method and derivatized for assay by glc-MS. From these experiments we were able to determine that the problem was associated with the final derivatization with rBDMS. EXPERIMENTAL Reagenrs. The tBDMS was obtained from two different suppliers, (1) a formulated tBDMS-imidazole in sealed ampoules from Applied Science Laboratories, and (2) crystalline tBDMS in pure form from Regis. Imidazole was from Matheson Coleman-Bell, dimethylformamide and 1-hexadecanol was from Eastman Organic Chemicals. The dimethylformamide was redistilled and dried over molecular sieve before using. Gus-liquid chromatography. A Varian model 2100 instrument was used for the initial screening runs, in which a rBDMS-hexadecanol reaction product was used to provide an immediate check on the formation of a tBDMS ether by each batch of reagent. A glass column 6 ft x 4mm i.d. was packed with 3% OV-1 on Chromosorb W AW 100-120 mesh and connected to the flame ionization detector (FID). Operating parameters were, nitrogen carrier gas 40 ml/mitt, injection port, oven, and detector temperatures at 230, 180, and 240°C respectively. FID range set at 10-I1 attenuation at 128X. 119 00062944/80/010119-03$02.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
120
SHORTCOMMUNICATIONS
A fresh batch of tBDMS-imidazole ampoules was obtained from Applied Science Laboratories, and the reagent transferred to a small Teflon-lined screw-cap vial under nitrogen and is referred to hereafter as Reagent A. Laboratory prepared solutions of the Regis tBDMS and the imidazole were made up in dry dimethylformamide (DMF) to the same final molar concentration as given by Applied Science with their ampoules, i.e., 1.0 mmole rBDMSlmilliliter and 2.5 mmole imidazolei milliliter. These solutions were used either as separate solutions to be mixed at the time of use (Reagent B) or as a premixed solution (Reagent C). All reagents were stored under nitrogen at - 12°C. 1-Hexadecanol was dissolved in dry DMF at a concentration of 0.1 mmole/milliliter and used to compare the reaction potency of the tBDMS reagent from both sources. A 20-~1 aliquot of hexadecanol solution was mixed with 20 ~1 of the reagent, or in the case of separate solutions, 10 ~1 each of tBDMS and imidazole. The reaction was done in small l-ml Teflon-lined screw-cap vials. The glc profiles were developed using l-w1 injections at zero reaction time and repeated at 24-hr reaction time at room temperature. Subsequent reaction mixtures were set up at various daily intervals to check the stability of the reagents. Having established optimum reaction conditions, urine samples containing the d3 PGF-M methyl ester “spike” were subjected to the extraction, purification, and derivatization procedure and assayed by glc-MS. RESULTS
Comparison of Reagent A versus Reagent B is shown in Fig. 1. Regis Lab formulated reagent produced a larger reaction product peak of tBDMS-hexadecanol derivative than did the Applied Science reagent. The locally formulated premixed Reagent C gave comparable results to Reagent B and “free” hexadecanol was absent with all three reagent reactions. Reassay of the same reaction mixtures at 24 hr, showed a diminished reaction product for both reagents A and B, with a corresponding increase of a secondary reaction peak at 1.5 min retention time. Stability of reagents A and B was good for several days, however, some unreacted hexadecanol was present using ‘I-day-old reagents. Reagent C (locally premixed) appeared to deteriorate much faster having no reaction potency at 7 days. The secondary reaction product peak at 1.5 min retention time was evident with all three reagents. It gradually increased in concentration in subsequent runs, as the reagents aged. The source of this peak appears to be a product of the tBDMS-imidazole mixture, since glc profiles of the two separate solutions even when 7 days old, showed no such peak. However, when mixed together and injected immediately, a peak did
121
SHORT COMMUNICATIONS
Id
FIG. 1. Gas-liquid chromatography traces of reaction product tBDMS-hexadecanol (retention time 11 mitt). (la) Reagent A (Applied Science) and (lb) Reagent B (Regis), immediate assay: (1~) Reagent A and (Id) Reagent B. repeat assay at 24 hr.
appear at the 1.5min retention time. No attempt was made to identify this secondary reaction product by glc-MS. Assay of the product of the “immediate reaction” using the spiked urine samples, instead of after an overnight reaction as originally described by Brash et al., yielded a 45-50% increase in reaction product as assayed with glc-MS. CONCLUSION
In conclusion, freshly prepared rBDMS-imidazole reagent is the most desirable one to use in terms of potency. Assay of *‘immediate reaction” samples result in higher yields of product from both hexadecanol and the prostaglandin F,a! metabolite. The Applied Science reagent in sealed ampoules give no formulation or expiration date and the lower yield may be due to aged reagents. ACKNOWLEDGMENTS We wish to express our thanks to Mr. R. W. Silverman for providing the glc-MS assays. Supported in part by USPHS-RR5354, American Heart Association LAS93, and a grant from Pharmaceutical Manufacturers Association Foundation.
REFERENCE 1. Brash, A. R., Baillie, T. A., Clam, R. A., and Draffan, G. A.,Eiochem. Med. 16,n (1976). KENNETH BRICKNELL MATTHEW E. CONOLLY
University of California at Los Angeles, School of Medicine, Division of Clinical Pharmacology, Department of Pharmacology, Los Angeles, California 90024 Received February 8, 1919