BIOLOGICAL MASS SPECTROMETRY, VOL. 20, 4 1 4 3 (1991)
Short Communication GC/MS/MS Detection of detomidine carboxylic acid in horse urine
Detomidine carboxylic acid is a major urinary metabolite in the horse of the veterinary analgesic sedative drug detomidine (1). This communication describes a rapid solid-phase extraction (SPE) method for this metabolite, followed by derivatisation and detection by GC/MS/MS. The use of tandem MS was necessary to overcome problems of co-elutants which could not be selectively removed during the extraction stage (reference 2 gives details of other approaches tried during method development). This method is currently in use in our laboratory for the analysis of equine urine, to detect the administration of detomidine. Using a Vac-ElutTM manifold (Jones Chromatography, Hengoed, UK), urine (3 ml, pH 3) is passed slowly under low vacuum ( 5 in Hg) down a Bond Elut Certify LRCTM SPE cartridge (Jones) which has been preconditioned with methanol (2 ml) followed by sodium phosphate buffer (2 ml, 0.1 M, pH 3). The cartridge is washed with acetic acid (1 ml, 1.0 M), then dried under full vacuum (15 - 20 in Hg, 5 min) The cartridge is washed sequentially with methanol (6 ml) , then ethyl acetate: ammonia solution, sp.gr. 0.88, (5 ml, 97:3 v/v) and dried (2 min) after each wash.
.
Detomidine carboxylic acid is eluted fromthe cartridge with butan2-01: ammonia solution (3 ml, 9:l v/v) and the eluate is dried under a stream of nitrogen at 8OOC. The residue is acetylated by heating (1 h, 8OOC) in ethyl acetate: acetic anhydride: acetic acid (300 p1, 4:l:l v/v). The solvent is removed under nitrogen at 8OoC, the residue redissolved in sodium sulphate-dried ethyl acetate (20 pl) plus MSTFA ( 5 pl) and an aliquot (1 pl) analysed by GC/MS/MS for the 0-TMS ether of the acetylated metabolite. The mass spectrometer used is a Finnigan MAT TSQ 70 triple quadrupole instrument, linked via a transfer line maintained at 28OOC to a Varian 3400 GC equipped with an OV1 fused silica capillary column (1 pm film thickness, 0.3 mm i.d., 8 m long). The sample is introduced v i a a split/splitless injector (24OoC, splitless mode, purge on at 0.6 min) . The oven temperature is held at 100°C for 1 min, then programmed from 100 to 3OOOC (20°C/min) and finally held at 3OO0C for 2 min. MS/MS analysis is carried out in the EI (70 eV) daughter ion (CAD, reagent gas argon) mode, scanning daughters of m/z 198 over the range m/z 80 - 200 in 1 s . The MS/MS conditions are optimised for the daughter to parent ion transition m/z 198 to 143 using the methyl ester of detomidine 3-carboxylic acid, which fragments to give the same abundant ion at m/z 198 as the acetyl/TMS derivative. The methyl ester, rather than the acetyl/TMS derivative, is used for tuning to avoid contamination; it is prepared by heating (30 min, 8OOC) detomidine 3-carboxylic acid in methanol: acetyl chloride (10:1 v/v)
.
Typical ion chromatograms from a post-detomidine administration urine extract are shown in Fig. 1. The mass spectrum obtained (arrowed peak, Fig. 1) is shown in Fig. 2a. This is identical to that of a derivatised standard (Fig. 2b), prepared by acetylating detomidine 3-carboxylic acid as described above and co-injecting with MSTFA. 1052-9306/91/01004143 $05.00
0 1991 by John Wiley & Sons, Ltd.
Received 21 September 1990 Revised 4 December 1990
SHORT COMMUNICATION
42
4:OO
6:OO
8:OO
1o:oo
retention time, min
Figure 1. Ion chromatograms of derivatfsed CertifyTM extract of post-detomidine administration horse urine.
Using this method, we have been able to detect detomidine carboxylic acid in post-detomidine administration urine samples for up to 23 h after a dose of 5 pg/kg, i.m. The metabolite has been detected after doses as low as 2.0 pg/kg, i.m.; experiments are under way at this laboratory to determine the lowest dose of detomidine after which the carboxylic acid metabolite is detectable, and for how long after dosing it remains so. The authors would like to thank Farmos Group Ltd., Turku, Finland for providing detomidine and detomidine 3-carboxylic acid for this work. M.A. Seymour, P. Teale and M.W. Horner, Horseracing Forensic Laboratory Limited, PO Box 15, Snailwell Road, Newmarket, Suffolk, CB8 7DT, UK. References 1. M.A. Seymour, P . Teale and M.W. Horner, Biorned. Environ. Mass Spectrorn., 19, 447, (1990).
Seymour, P. Teale and M.W. Horner, in M e t h o d o l o g i c a l S u r v e y s in Biochemistry and A n a l y s i s , vol. 2 0 , ed. by E. Reid and I . D . Wilson, p . 309. Royal Society of Chemistry, Cambridge, UK. (1990).
2. M.A.
SHORT COMMUNICATION
a
11
43
143
198
183
169
I
120
80
160
200
rnlz
198
0
143
183 c-0 I GH 3
u 155
116
0
3
00
120
160
I
I
200
rnlz
Figure 2.
Mass spectra (daughters of m/z 198.1) of a) derivatised Certifym extract of post-detomidine administration horse urine (arrowed peak, Fig. 1) and b) TM8 ester of acetyl detomidine 3-carboxylic acid.
Short Communication GC/MS/MS Detection of detomidine carboxylic acid in horse urine
Detomidine carboxylic acid is a major urinary metabolite in the horse of the veterinary analgesic sedative drug detomidine (1). This communication describes a rapid solid-phase extraction (SPE) method for this metabolite, followed by derivatisation and detection by GC/MS/MS. The use of tandem MS was necessary to overcome problems of co-elutants which could not be selectively removed during the extraction stage (reference 2 gives details of other approaches tried during method development). This method is currently in use in our laboratory for the analysis of equine urine, to detect the administration of detomidine. Using a Vac-ElutTM manifold (Jones Chromatography, Hengoed, UK), urine (3 ml, pH 3) is passed slowly under low vacuum ( 5 in Hg) down a Bond Elut Certify LRCTM SPE cartridge (Jones) which has been preconditioned with methanol (2 ml) followed by sodium phosphate buffer (2 ml, 0.1 M, pH 3). The cartridge is washed with acetic acid (1 ml, 1.0 M), then dried under full vacuum (15 - 20 in Hg, 5 min) The cartridge is washed sequentially with methanol (6 ml) , then ethyl acetate: ammonia solution, sp.gr. 0.88, (5 ml, 97:3 v/v) and dried (2 min) after each wash.
.
Detomidine carboxylic acid is eluted fromthe cartridge with butan2-01: ammonia solution (3 ml, 9:l v/v) and the eluate is dried under a stream of nitrogen at 8OOC. The residue is acetylated by heating (1 h, 8OOC) in ethyl acetate: acetic anhydride: acetic acid (300 p1, 4:l:l v/v). The solvent is removed under nitrogen at 8OoC, the residue redissolved in sodium sulphate-dried ethyl acetate (20 pl) plus MSTFA ( 5 pl) and an aliquot (1 pl) analysed by GC/MS/MS for the 0-TMS ether of the acetylated metabolite. The mass spectrometer used is a Finnigan MAT TSQ 70 triple quadrupole instrument, linked via a transfer line maintained at 28OOC to a Varian 3400 GC equipped with an OV1 fused silica capillary column (1 pm film thickness, 0.3 mm i.d., 8 m long). The sample is introduced v i a a split/splitless injector (24OoC, splitless mode, purge on at 0.6 min) . The oven temperature is held at 100°C for 1 min, then programmed from 100 to 3OOOC (20°C/min) and finally held at 3OO0C for 2 min. MS/MS analysis is carried out in the EI (70 eV) daughter ion (CAD, reagent gas argon) mode, scanning daughters of m/z 198 over the range m/z 80 - 200 in 1 s . The MS/MS conditions are optimised for the daughter to parent ion transition m/z 198 to 143 using the methyl ester of detomidine 3-carboxylic acid, which fragments to give the same abundant ion at m/z 198 as the acetyl/TMS derivative. The methyl ester, rather than the acetyl/TMS derivative, is used for tuning to avoid contamination; it is prepared by heating (30 min, 8OOC) detomidine 3-carboxylic acid in methanol: acetyl chloride (10:1 v/v)
.
Typical ion chromatograms from a post-detomidine administration urine extract are shown in Fig. 1. The mass spectrum obtained (arrowed peak, Fig. 1) is shown in Fig. 2a. This is identical to that of a derivatised standard (Fig. 2b), prepared by acetylating detomidine 3-carboxylic acid as described above and co-injecting with MSTFA. 1052-9306/91/01004143 $05.00
0 1991 by John Wiley & Sons, Ltd.
Received 21 September 1990 Revised 4 December 1990
SHORT COMMUNICATION
42
4:OO
6:OO
8:OO
1o:oo
retention time, min
Figure 1. Ion chromatograms of derivatfsed CertifyTM extract of post-detomidine administration horse urine.
Using this method, we have been able to detect detomidine carboxylic acid in post-detomidine administration urine samples for up to 23 h after a dose of 5 pg/kg, i.m. The metabolite has been detected after doses as low as 2.0 pg/kg, i.m.; experiments are under way at this laboratory to determine the lowest dose of detomidine after which the carboxylic acid metabolite is detectable, and for how long after dosing it remains so. The authors would like to thank Farmos Group Ltd., Turku, Finland for providing detomidine and detomidine 3-carboxylic acid for this work. M.A. Seymour, P. Teale and M.W. Horner, Horseracing Forensic Laboratory Limited, PO Box 15, Snailwell Road, Newmarket, Suffolk, CB8 7DT, UK. References 1. M.A. Seymour, P . Teale and M.W. Horner, Biorned. Environ. Mass Spectrorn., 19, 447, (1990).
Seymour, P. Teale and M.W. Horner, in M e t h o d o l o g i c a l S u r v e y s in Biochemistry and A n a l y s i s , vol. 2 0 , ed. by E. Reid and I . D . Wilson, p . 309. Royal Society of Chemistry, Cambridge, UK. (1990).
2. M.A.
SHORT COMMUNICATION
a
11
43
143
198
183
169
I
120
80
160
200
rnlz
198
0
143
183 c-0 I GH 3
u 155
116
0
3
00
120
160
I
I
200
rnlz
Figure 2.
Mass spectra (daughters of m/z 198.1) of a) derivatised Certifym extract of post-detomidine administration horse urine (arrowed peak, Fig. 1) and b) TM8 ester of acetyl detomidine 3-carboxylic acid.
