Anal. Chem. 1980, 52, 1815-1820
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Thin-Layer Chromatographic and High Resolution Mass Spectrometric Determination of p-Hydroxyphenylethylamines in Tissues as Dansyl-Acetyl Derivatives David A. Durden,” August0 V. Juorio, and Bruce A. Davis Psychiatric Research Division, 508A University Hospital, Saskatoon, Saskatchewan. Canada S7N OXO
A method has been developed for the quantitation in tissue of the biogenic P-hydroxyphenylethylaminesas their mixed dimethylaminonaphthalenesulfonyl-acetyl derivatives. The amines are derlvatlred, Isolated by thin-layer chromatography, and quantitated by high resolution mass spectrometry using deuterated analogues as internal standards. As little as 1 or 2 picomoles (200 pg) of the amine may be quantitated in tissue samples. The sensitivity and specificity of the method have been demonstrated through the analysis of some representative neurologlcal tissues for m- and p-synephrine which have been shown for the first time to be present in animal tissue. The method is more sensitive and specific than previous chemical methods.
T h e use of t h e dimethylaminonaphthalene-5-sulfonyl (dansyl) derivative and high resolution mass spectrometry have been successfully applied to the quantitation of t h e following arylalkylamines in neural tissue and body fluids: phenylethylamine, m- and p-tyramine, tryptamine, benzylamine, amphetamine, p-hydroxyamphetamine, and adrenaline to (1). I t was observed previously (2) that as little as mol of these amines could be detected in the mass spectrometer. However, determination of most of the @-hydroxyphenylalkylamines as their dansyl derivatives was not successful as the presence of the free @-hydroxygroup caused t h e molecular ion to give a nonlinear signal in the mass spectrometer and limited the minimum detectable level to between and mol. Acetylation of the @-hydroxy group has resolved this deficiency. Methods that have previously been successfully applied to t h e analysis of @-hydroxyphenylethylaminesin tissue are radioenzymatic assays for phenylethanolamine, m- and p-octopamine, noradrenaline, and adrenaline (3-14), gas chromatography-mass spectrometry for phenylethanolamine, octopamine, metanephrine, normetanephrine, noradrenaline, and adrenaline (15-20), and fluorimetry for noradrenaline and adrenaline (21-23). The radioenzymatic methods are capable of detecting as little as 10 pg (100 fmol) of an amine, but they have some limitations. The enzyme may be inhibited by salts such as calcium (12) or other endogenous compounds or drugs present in t h e tissue extracts (13, 14) as substrates. The radioenzymatic methods have not been used for N-methylphenylethanolamine, m- or p-synephrine, or metanephrine and normetanephrine. T h e gas chromatography/mass spectrometric (GC/MS) method has a sensitivity of the same order as the high resolution mass spectrometric method. At present it has not been employed for t h e synephrines nor has the chromatography been capable of separating the meta and para isomers. T h e fluorimetric method is less sensitive than the radioenzymatic assay and has only been used for quantitation of t h e catecholamines in tissue. The present high resolution mass spectrometric method was developed to avoid some of the problems associated with the
enzymatic assay and to extend the number of compounds analyzed over those of the GC/MS method. As an example of the use of the method, we report here the quantitation of m- or p-synephrine individually and together (as “total” synephrine) in bovine and mouse adrenal glands.
EXPERIMENTAL Materials. All solvents were glass distilled or purchased as “HPLC” grade. Water was deionized and glass distilled. Dansyl chloride (Aldrich,Milwaukee, Wis.) WEB purchased from Terochem Laboratories, Edmonton, Canada, and silica thin-layer plates (E. Merck, Darmstadt, Germany) from Brinkmann Canada Ltd., Rexdale, Ontario. The amines were purchased as follows: phenylethanolamine, Aldrich Chemical Co., Milwaukee, Wis.; rnoctopamine (norphenylephrine) and p-octopamine, Sigma Chemical Co., St. Louis, Mo.; rn-synephrine, Wyeth Laboratories, Westchester, Pa.; p-synephrine, Regis Chemical Co., Morton Grove, Ill.; normetanephrine, metanephrine, and noradrenaline (DL-arterenOl) Calbiochem., La Jolla, Calif., and DL-adrenaline, Light & Co., Colnbrooke, England. The deuterated amines, 1phenyl-2-aminoethanol-1,2,2-d3, DL-normetanephrine-c,a-d2,P-dl hydrochloride noradrenaline d3 (norepinephrine-a,a-d2,/3-dlhydrochloride), and adrenaline (DL-epinephrine-a,a-d2~-d~) were purchased from Merck, Sharp and Dohme, Canada Ltd., Point Claire, Quebec, Canada. The other amines were synthesized as described below. Synthesis of N-Methylphenylethanolamine.w-Bromoacetophenone, prepared from acetophenone and bromine in ethanol, was added to a solution of N-methylbenzylamine in ether, the precipitate was filtered, and the filtrate was washed, dried, and evaporated to give w- [N-benzyl-N-methylamino]acetophenone. The keto group of the latter was reduced with lithium aluminum hydride in ether, then debenzylated by hydrogenation at 30 psi hydrogen over 10% palladium on charcoal. The product (mp 103-104 “C) was precipitated from ether as the hydrochloride and purified by trituration in ethyl acetate and acetone. Synthesis of p-Synephrine-d3. co-Bromo-p-hydroxyacetophenone was prepared by the addition of a chloroform solution of p-hydroxyacetophenone to a suspension of cupric bromide in ethyl acetate. After 2-h refluxing, the solvents were evaporated and the residue was treated with ice-cold hydrochloric acid. The product was filtered, dried, and dissolved in the minimum of ethanol, then slowly added to a stirred solution of two equivalents of N-methylbenzylamine in dry ether. The precipitated Nmethylbenzylamine hydrobromide was filtered, the product was isolated from the filtrate, and two deuterium atoms were incorporated in the side chain by heating with deuterium oxide/Odeuteroethanol. Three such exchanges were carried out. Reduction of the keto group and cleavage of the benzyl group were achieved simultaneously by hydrogenolysis of the hydrochloride using 10% palladium on charcoal in deuterium oxide/Odeuteroacetic acid at 40 psi deuterium gas to produce p-synephrine-d3,mp 152-153 “C. Reduction of the keto group requires that the palladium on charcoal be freshly prepared immediately before use and that it be thoroughly washed and dried before use. Deuterium incorporation as determined mass spectrometrically on the bis-dansyl derivative was: do = 0.5%, dl = 2.070, d2 = 11.170, d 3 = 68.7%, d4 = 12.6%, d 5 = 3.190,and d6 = 2.090. Synthesis of rn-Synephrine-d3, The procedure was the same as for p-synephrine-d,, using rn-hydroxyacetophenone as the starting material.
0003-2700/80/0352-18 15$01.00/0 0 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
Synthesis of p-Octopamine-d,. p-Benzyloxy-o-bromoacetophenone was prepared from p-hydroxyacetophenone by benzylation followed by bromination with bromine in refluxing benzene. The product was converted to the azide by gentle refluxing with sodium azide in methanol and then the a-hydrogen atoms were exchanged with deuterium by refluxing in 0-deuteromethanol; three such exchanges were carried out. The product was reduced with lithium aluminum deuteride in ether and then the hydrochloride was debenzylated by hydrogenation over 10% palladium on charcoal. The melting point was 180-182 "C. Deuterium incorporation was determined mass spectrometrically on the dansyl derivatives: dl = 3.1%, d2 = 17.6%, d3 = 54.6%, d4 = 18.0%, db = 3.9%, d,j = 1.7%, d7 = 1.1%. Synthesis of m-Octopamine-d,. The procedure was the same as for p-octopamine-d,, using m-hydroxyacetophenone as the starting material. Synthesis of the Dansyl Amines. About 25 mg of the amine was dissolved in 10 mL of water. The solution was saturated with sodium carbonate, 20 mL of acetone containing 1-5 mol equiv of dansyl chloride were added, and the mixed solution was left to react overnight in the dark at room temperature. The acetone was evaporated in a stream of nitrogen and the dansyl amine extracted into 20 mL of benzene and recrystallized from ethanol/water. The reaction was repeated for the catecholamines to ensure complete derivatization. The amines were examined by thin-layer chromatography (TLC) and mass spectroscopy to verify purity. Synthesis of Dansyl Acetyl Amines. The dansyl amine product from the above reaction was acetylated in 10 mL of benzenepyridine-acetic anhydride ( 1 O : l : l ) for 1 h a t room temperature. A portion of the product was separated by TLC and examined for the presence of nonacetylated amine, or N-acetylated amine. The solution was washed (4 X 10 mL) with water and the dansyl-acetyl amines were recrystallized from ethanol/water solution. Again, thin-layer chromatography was performed and the zones were examined mass spectrometrically. Purity of the Deuterated Amines. The deuterated amines were recrystallized to a constant mp value which was compared with the published mp of the nondeuterated analogue. The mass spectra of the amine as the HC1 salt and as its dansyl derivative were examined and the deuterium content was determined after making appropriate corrections, for 13C,lSO,and 34S. Before use as an internal standard in tissue experiments, the purify factor F of each deuterated compound was obtained from the slope of a calibration curve determined mass spectrometrically from the molecular ion intensities of varying amounts (1to 50 ng) of normal amine (primary standard) and constant amounts (50 or 100 ng) of the deuterated amine after derivatization and thin-layer separation. Methods. Mass Spectrometry. Mass spectra (Figure 1)were recorded using an A E I MS902S double focusing mass spectrometer equipped with a direct insertion probe. The fixed length probe used disposable sample cups prepared from borosilicate melting point capillaries and the length was adjusted to give maximum signal (2). The spectra were recorded at a resolution of 1000 with an electron energy of 70 eV and ion source temperatures from 225 to 300 "C and were processed by a VG 2050 F / B data system. For quantitative purposes integrated ion currents of the molecular ions were measured a t 7000 resolution a t temperatures from 250 to 325 "C. The signals were integrated using a Digital Equipment Lab 8/e system programmed in BASIC ( 1 ) . The dansyl derivatives and the dansyl-acetyl derivatives were examined for linearity of detection of the mass spectrometer by to lo4 mol amounts of the amines from the probe evaporating and the minimum detectable level was measured with the electron multiplier gain set a t 1 X lo7 (3000 V). The linear range, minimum detectability and mass spectrometer operating data are listed in Table I. The amount of amine in the sample was calculated from: ng =
AiowX W Ahigh -
c
X
F (1)
Alow
where Alowand Ahighare the integrated ion currents of the low mass ion (endogenous amine) and the high mass ion (d, internal standard), W is the amount of deuterated internal standard added
to the homogenate, and F is the purity factor corresponding to the isotopic fraction that was the d3 species (0.85 to 1.0). The factor C is to correct for the M + 3 ions associated with the low mass molecular ion due to the %, 13C,ISO, and 15Nisotopes. These ions form a composite ion which has a mass only a few parts per million different from the ion due to the d 3 labeled amine and it is not separable a t the mass spectrometric resolution (7000 or 10000) used in these analyses. The blanks were calculated in nanograms and an average value was subtracted from the sample values. The minimum practical detectable level (Table I) was taken as the value which gave a signal twice the blank value and represents the minimum sample that can usually be detected by the analytical method. Decomposition of the bis-dansyl derivatives of octopamine and synephrine during evaporation of the sample from the probe tip (Figure 2) was measured by comparing the intensity ratio of (M - 2)+/M+ [or (M - 3)+/M+ for the d 3 amines]. Quantitation of P-Hydroxyamines in Tissue. After dissection, tissues were frozen on dry ice, weighed and homogenized in 0.1 N hydrochloric acid containing ascorbic acid (5 mg/mL) and EDTA (1mg/mL). Appropriate quantities of the deuterated internal standard (25 to 100 ng) were added to the homogenate so that the amount added exceeded the amount of amine expected to be found in the sample. The homogenate was saturated with sodium carbonate, frozen and thawed twice, centrifuged, and transferred to a test tube (100 X 16 mm) and then two volumes of acetone containing 16 mg of dansyl chloride were added to the supernatant. The mixture was allowed to react overnight at room temperature in the dark. The excess sodium carbonate was precipitated by the addition of 2 mL of benzene and the organic layer was transferred to a new test tube and evaporated to dryness under a stream of nitrogen in a water bath a t 40 to 45 "C. The extract was redissolved and acetylated in 2 mL of ethyl acetate-pyridine-acetic anhydride ( l O : l : l , v/v) for 1 h a t room temperature in the dark. The mixture was then evaporated to dryness under nitrogen, redissolved in 0.2 mL of toluene, and transferred to a 20 X 20 cm silica gel thin-layer plate in a 1.5-cm streak. A second 0.2 mL of toluene was used to rinse the tube and also applied to the plate. Six samples and a reference standard could be accommodated on each plate. The plate was then developed in one of the first solvent systems (Table 11) and visualized under UV a t 254 nm. Usually only one amine was quantitated per experiment. The zone containing the dansyl-acetyl-amine was scraped from the plate and eluted with ethyl acetate. This extract was then dried under nitrogen, redissolved in 0.2 mL of toluene and applied to a second plate which was developed in the second solvent system. If required, the amine was isolated on a third plate. After the last chromatogram, the derivatives were extracted into 20 to 25 pL of ethyl acetate in a constricted mp capillary in preparation for mass spectrometric analysis as previously described (24). Blanks were obtained by subjecting 0.1 N HC1 samples to the procedure including addition of the deuterated amine, dansylation, acetylation, chromatographic isolation, and mass spectrometric analysis. Similarly the linearity of the method was determined by processing standards containing known amounts of the amine. It is possible to isolate each of the amines except the meta and para isomers of octopamine or synephrine which are co-chromatographic and are detected to give total octopamine or total synephrine. Solvent systems and R, values are given in Table 11. This method was used to determine together m- and p-synephrine as "total" synephrine in mouse hypothalamus, heart, and adrenal, and bovine adrenal tissue (Table 111) using tri-deutero-p-synephrine as the internal standard. Determination of meta a n d para Isomers of Octopamine a n d Synephrine. Since it was found possible to separate the meta and para isomers of these amines as their bis-dansyl derivatives, the procedure was modified. The above method was followed to the point that the bis-dansyl derivatives had been prepared and extracted into benzene. The benzene solution was evaporated t o dryness and the residue (containing the bis-dansyl derivatives of octopamine and synephrine) was redissolved in 0.1 mL of toluene and applied to a thin-layer plate and developed in the solvent system chloroform-butyl acetate (5:3 v/v). The R, of the m- and p-octopamine zone was 0.20 to 0.22 and that of the rn- and p-synephrine zone was 0.35 to 0.39. The two zones were scraped, eluted, evaporated, and applied to separate thin-
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
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ANALYTICAL CHEMISTRY, VOL.
52, NO. 12, OCTOBER 1980
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layer plates and developed in the system benzene-triethylamine (5:2 v/v). The meta and para isomers of the bis-dansyl amines were separated on these plates with the following R, values: p-octopamine, 0.18; m-octopamine, 0.22; p-synephrine, 0.44; and rn-synephrine, 0.48. These zones were scraped into separate test tubes. Each amine was eluted with ethyl acetate and acetylated in 1mL of ethyl acetatepyridineacetic anhydride as above. The bis-dansyl-acetyl-octopamines and synephrines were isolated from other reaction products by a third thin-layer separation in the system chloroform-ethyl acetate (6:lv/v) and eluted with ethyl acetate in preparation for mass spectrometry. Cross contamination of the meta and para zones was determined using mixtures of p-amine and rn-amine-d3or m-amine and p-amine-d3,for both octopamine and synephrine. This method was used to determine concentrations of m- and p-synephrine in mouse and bovine adrenals (Table 111). RESULTS AND DISCUSSION Chromatography. The chromatographic systems in Table 11, for the separation of the P-hydroxyphenylethyl amines as the dansyl-acetyl derivatives, are designed so that one or two
amines to be quantitated in a particular experiment are isolated and all other amines are discarded. We did not try to isolate all amines simultaneously, as the required compromises reduce the purity of each zone. The solvent systems for octopamine and synephrine do not separate the meta and para isomers from each other. Other pairs of amines which were particularly difficult to separate were metanephrine and adrenaline and normetanephrine and noradrenaline. However, the molecular weights of these amines are considerably different and the fragmentation pattern of the larger molecule is such that it does not interfer with quantitation of the lower weight amine. Considerable effort was made to separate the meta and para isomers of octopamine and synephrine as the dansyl-acetyl derivatives. None were particularly successful. However, these amines were readily separated as the bis-dansyl derivatives, which contain the P-hydroxyl group with a free proton. Consequently, it was decided to isolate the octopamines and synephrines as bis-dansyl derivatives and then acetylate. We
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
attempted to acetylate the bis-dansyl derivatives in the melting point capillaries, by eluting the amines from the silica gel into 25 p L of ethyl acetate-acetic anhydride-pyridine lO:l:l, but found that the recovery was not as high as that obtained when the amines were acetylated in a larger volume. T h e pyridine did not evaporate from the mass spectrometer direct probe tip as readily as ethyl acetate, and suppression of the octopamine or synephrine signal occurred during the ion current integration due to co-evaporation of the large amounts of solvent residues. Isolation of the dansyl-acetyl derivative in a third chromatographic system removed these impurities. Since we were using deuterated internal standards, no attempt was made to remove the zone quantitatively and it was estimated that the deuterium standards were recovered a t between 10 and 20%. Instead, only the most intensely fluorescent region of the zone was taken and, although the yield was reduced somewhat, the specificity and the ability t o separate isomers were improved considerably. T o obtain the separations reported here, it is essential that the silica gel thin-layer plates be reactivated before use. Spectra. In the spectra of the dansyl-acetyl P-hydroxyphenylethylamines reported in Figure 1, the base peak mle 170 and the prominent ions m l e 234 or 235, and the (M 233)’ or (M - 466)’ ions are due to the dimethylaminonaphthalene ion, the dimethylaminonaphthalenesulfonylion, or loss of one or more dansyl groups from the molecular ion as previously reported for dansyl compounds (2). The spectra contained prominent ions due to cleavage of the alkyl chain carbon bonds (with or without rearrangement of one hydrogen atom) and ions due to the acetyl group ( m l e 42) or its loss from the molecular ion as (M 60)’. In all the spectra, except t h a t of noradrenaline, the molecular ion is very prominent,, Le., 5 to 30% of the base peak intensity, and is the ion most suitable for quantitative analysis due to its specificity. A comparison of each spectrum of the dansyl-acetyl amine with t h a t of the dansyl amine [previously reported were those of p-octopamine, normetanephrine, metanephrine ( 2 ) and adrenaline ( 2 5 ) ]shows t h a t in all cases the molecular ion has the greater intensity in the spectrum of the acetylated derivative. We have previously reported that in the spectra of the bis-dansyl derivatives of m- and p-synephrine that loss of H, (or HD in the tri-deuterated species) from the molecular ion occurred when sub-nanomolar amounts of the derivative were evaporated from the direct probe (26). We also observed this phenomenon when examining the spectra of bis-dansyl m- and p-octopamine. Acetylation of the /3-hydroxy group eliminates this loss. Small differences in the spectra of the meta and para isomers of octopamine and synephrine are detectable by examining the intensities of the “mono dansyl” (M - 233)’ ion, This ion is slightly more intense in the spectra of the dansyl acetyl derivatives of the meta isomers. However, it probably cannot be used as an unequivocable determinant of the isomeric structure since the fragmentation changes somewhat depending upon the amount of sample evaporated from the probe. Consequently we used the chromatographic data to identify the isomers. Integrated Ion Current Data. Table I shows the data obtained when various amounts of the dansyl or dansyl-acetyl derivatives of the amines were evaporated from the direct probe and the ion current is integrated. T h e relative sensitivity was obtained by comparison of the computer integrated signals ( I ) (equivalent to the peak area on the UV recorder) of equimolar amounts (lo-’’ mol) of the amine derivative and dansyl-phenylethylamine which we have previously used as a standard ( 2 ) . T h e minimum amount detected was that amount which gave a signal of twice the base-line noise a t a resolution of 7000. T h e linear range is the range of mole ~
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quantities which gave a signal in the mass spectrometer proportional to the amount evaporated from the direct probe tip. An ion from the mass reference compound, either heptacosafluorotri-n-butylamineor perfluorokerosene, was used to verify the exact mass of the observed ion and as an external intensity reference. At the high end of the range, the intensity of this ion, along with the signal due to the evaporated amine derivative, decreased due to suppression of the ion signal by too large a sample concentration. T h e minimum practical detectable amount is the amount of amine which when added to the amount of tri-deutero amine internal standard (Table I) gave a signal twice the blank. The samples, in this case, were treated in the same manner as tissues, i.e., derivatized, separated by thin-layer chromatography, and eluted before mass spectrometric analysis. T h e greater relative sensitivity of the dansyl-acetyl derivative compared to the dansyl derivatives appears to be due primarily to the greater number of ions remaining as the molecular ion. For all the amines except phenylethanolamine and N-methylphenylethanolamine, the minimum amount detected by the mass spectrometer is lower for the dansylacetyl derivatives, in part due to the increased size of the molecular ion, but mainly due to the reduced amount of decomposition of the small amounts to mol) during evaporation from the probe tip. Likewise the linear range is wider (by a factor of 10) for most of the corresponding acetylated derivatives. T h e major increase in sensitivity of the analytical method, as indicated by the minimum practical level, for octopamine and synephrine is due to the prevention of loss of hydrogen (as M - HD) from the trideuterated molecular ion. Thus for most of the amines, the method is capable of quantitating amine levels in tissues down to about 200 pg. Deuterated Internal Standards. We found that the melting points of most of the deuterated amines were within one or two degrees of those published for the unlabeled analogues. The exceptions were m- or p-octopamine-d3 which melted sharply seven t o eight degrees above those reported for the unlabeled amines. Since it was not easily possible to synthesize the amines t o be 100% as the tri-deutero compound, we decided t o use them as secondary standards. A second reason for utilizing them as secondary standards was that we observed t h a t the molecular ion of the tri-deutero compound was a different fraction of the total ionization (usually slightly greater) than was the molecular ion of the unlabeled amine. This slightly affected the relative sensitivity of the mass spectrometer to the two amine derivatives when quantitating using the integrated ion current procedure. Thus the factor F (Equation 1) is a composite of the chemical purity, the isotopic purity of the deuterated amine, and the relative sensitivity of the mass spectrometer to the two isotopic species. For the precision obtainable in determining amine levels in biological samples, this method provided more than adequate accuracy. Loss of H, from Bis-dansyl Octopamine or Bis-dansyl Synephrine. The dehydrogenation of the bis-dansyl derivatives of octopamine and synephrine was studied in an attempt to determine whether this occurred during chromatography, during evaporation from the direct probe or during the ion fragmentation process. Figure 2 shows the variation of the ratio of the M - H, ion to the molecular ion t o the amount of sample evaporated from the direct probe. I t appears that this process occurs a t approximately the same rate for the protonated and the deuterated amines. The increase in decomposition t o form the (M - H,)’ion was observed as a concurrent increase in the relative size of this ion as lower concentrations of solutions containing the amine were applied to the probe tip and was not a mathematical artifact due to
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ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 12, OCTOBER 1980
Table 111. Concentrations of Synephrines in Various Representative Tissues tissue mouseC hypothalamus moused heart froge heart mouse adrenal Lovine adrenal
weight, mg
“total” synephrine, ngiga
rn-synephrine, nglg
56 i 9 (3) 230 i 7 0 ( 4 ) 140 i 2 0 ( 6 ) 6.6 f 0.5 (8) 11.7 i 0.7 ( 1 0 )
