182
Anal. Chem. 1982, 5 4 , 182-186
Optimization of a Paired-Ion Reversed-Phase Liquid Chromatographic Separation of Synthetic Phencyclidine Mixtures LOUISA. Jones,* Rodney W. Beaver, and T. L. Schmoeger Department of Chemistry, North Carolina State Universlty, Raleigh, North Carolina 27650
A method Is presented for the optlmlxatlon of the reversedphase lon-palr LC separatlon of phencyclldlne (PCP) synthetlc mlxtures. Combined effects of column type, solvent composnlon and palrlng Ion (chaln length and concentration) on log k’ for PCP (k’])and I-[%( 1,l‘-blphenyl-Cyl)cyclohexyl]plperldlne (k’II) as well as qIII were evaluated by modlfled “MAPS” and two-dlmenslonal plots. The latter method, utillzlng aII,I vs. % MeOH with constant palrlng-Ion chaln length, proved to be as effective as the “MAPS” approach In 88lectlng optlmum separation parameters for I and I1 whlch resulted In Improved separatlon for the 1I peaks observed. The resultlng optlmlred system employs a C-18 column and a moblle phase of 70% methanol In water with the hexanesulfonate palrlng Ion and Is demonstrated on PCP synthetlc mlxtures contalnlng four ldentlfled compounds and seven unldentlfled compounds.
Phencyclidine, 1-(1-phenylcyclohexy1)piperidine(PCP, I), a dissociative analgesic of severe social abuse, is illicitly prepared by the method of Kalir ( I ) to produce the compound of interest in 65-85% yield (2). The determination of PCP in seized illicit preparations, “pure” street drug seizures, or in “doped” cigarettes, has been the major thrust of published methods (3-6) which have been devoted to establishing (qualitatively) the presence of PCP. These methods, however, have not examined impure PCP preparations for compounds other than the principal ingredient. We became interested in determining other PCP-like compounds cosynthesized in the Kalir (I) method and used, as our investigatory sample, the extractable bases of this preparation. Since GC methods were found to give rise to suspected thermal decompositions (5),our attention was directed to the development of a liquid chromatography (LC) system suitable for the isolated bases. In preliminary investigations, both normal-phase (hexane/chloroform and chloroform/ methanol on silica) and reversed-phase (methanol/water, acetronitrile/water, and THF/water, on (2-18and CN columns) methods proved unsuitable due to severely tailing peaks. Since the basic nitrogen of the piperidine ring in PCP and its cosynthetics can interact with active sites in the column, the poor peak symmetry observed was attributed to hydrophobic and adsorption mechanisms operating simultaneously (7)which gave rise to nonlinear adsorption isotherms. The addition of a pairing agent, heptanesulfonate, produced a dramatic improvement in peak symmetry and prompted us to investigate in depth the effect of pairing agents, column types, and solvent composition on the separation of PCP mixtures. A modification of the method reported by Sachok et al. (8)was used to optimize the separation of PCP (I) and its cosynthetic 1-[ 1-(1,l’-biphenyl-4-yl)cyclohexyl]piperidine (11). Although Lurie and Demchuck (9) reported the effects of methanol/water/alkylsulfonate(CH3-, n-C4H9-, n-C7H,3-) on the retention characteristics of a variety of nitrogen-con-
taining compounds including PCP, their system was not designed for the analysis of synthetic PCP mixtures. The analytical method developed here illustrates the separation of some of the cosynthetics identified in the mixtures, which included 1-[l-(l,l’-biphenyl-4-yl)cyclohexyl]piperidine(II), 1-[1-(phenylethyl)cyclohexyl]piperidine(111), and 1,l’-(1,4phenylenedicyclohexylidine)bis[piperidine] (IV) (IO). (See Figure 1 for structures.)
EXPERIMENTAL SECTION Apparatus. A Waters Associates (Milford, MA) ALC/GPC 244 liquid chromatographwas used for all analyses. Mobile phase delivery was accomplished by twin M6000A pumps interfaced through the Waters Model 660 solvent programmer. Detection was by UV (Waters Model 440) at 254 nm at a sensitivity of 0.02 AUFS and the chromatogramswere obtained by using a Houston Instruments Omniscribe strip chart recorder (Houston TX). Columns examined included Waters pBondapaks CN and C-18 (both 3.9 mm i.d. X 30 cm) and a Brownlee RP-8 (Santa Clara, CA, 4.6 mm i.d. X 25 cm). Reagents and Samples. Methanol (MeOH) and acetonitrile (MeCN) were LC grade and obtained from Fisher Scientific (Fairlawn, NJ). Water was deionized and was passed through a 100 cm X 4.6 mm i.d. column packed with Bondapak C-l8/ Porasil B (Waters Associates). The sodium salts of pentanesulfonate (C5S03Na),hexanesulfonate (C6S03Na),and octanesulfonate (C&OJ’Ja) and methanesulfonic acid were obtained from Fisher Scientific,as was reagent grade acetic acid (HAC). Crude PCP was synthesized according to the method of Kalir ( I , IO). Solvent Preparation. Solvents containing 5.0 mM C5S03Na, C6S03Na,and C8S03Nawere all prepared in a manner similar to C8SO9Na: To 1000.0 mL of either MeOH or MeCN 40.0 mL of HzOwas added followed by 25.0 mL of glacial acetic acid (HAC), and 0.941 g (5.0 mmol) of C6S03Na;the solution was then filtered through a 0.5-pm Fluoropore filter (Millipore Corp., Bedford, MA). The aqueous portion of the mobile phase was prepared by adding 25.0 mL of glacial HACplus 0.941 g of C&O3Na to 1000.0 mL of HzO and filtering through a 0.45-pm cellulose acetate filter (Millipore Corp.) which gave a solution of pH 3.5. Methanesulfonate was not available as the sodium salt, so a slightly different procedure was used to prepare mobile phases containing CH3S03Na. A 500-mL portion of 0.125 M CH3S03H in HzOwas carefully titrated to pH 5-7 with concentrated NaOH. A 40.0-mL sample of this solution was added to 1000.0 mL of MeOH or MeCN along with 25.0 mL of HACwhich gave a solution equivalent to that which would be obtained by adding 5.0 mmol of CH3S03Na+ 40 mL of HzOto 1000.0 mL of organic solvent. Aqueous solutions of CH3S03Nawere obtained by adding HzO to 40.0 mL of the 0.125 M CH3S03Nasolution along with 25.0 mL of glacial HACto make 1000.0 mL, the pH of this solution being 3.5. The effects of varying concentration of hexanesulfonatewere examined with solutions prepared as above but which contained C6S03Naat concentrations of 2.5 or 7.5 mM. Analysis and Calculations. The standard solution used for the determination of k’and the selectivity ratio, a,was 0.30 pg/pL of crude PCP in MeOH. Each injection was 10.0 pL at a mobile phase flow rate of 2.5 mL/min. Column void times (to)were obtained by injection of pure MeOH or HzO. The k’values for PCP (I) and (11) and aIIII(the selectivity between I and 11; peaks I and 11, Figure 6) were calculated in the usual manner (11).
0003-2700/82/0354-0182$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54,
NO. 2, FEBRUARY 1982
Table I. Least-Squares Equationsa for log k‘I vs. % MeOH and log k‘I vs. o/r MeCN in Mobile Phase MeOH/H,O MeCN/H,O C-8 (2-18 CN C-8 (2-18 pairing ion slope/intercept slope/intercept slope/intercept slope/intercept slope/intercept CH,SO,Na C,SO,Na C,SO,Na C,SO,Na
-0.0296/1.94 -0.0309/2.24 -0.0332/2.45 -0.0398/3.01
-0.0352/2.16 -0.0348/2.15 -0.0401/2.64 -0.0483/3,51
-0.0143/0.667 -0.0145/0.709 -0.0152/0.796 -0.0173/0.997
-0.0212/1.51 -0.0214/1.68 -0.0220/1.63 -0.0221/1.69
-0.0170/0.952 -0.0405/1.86 -0.0336/1.72 -0.0263/1.45
183
CN slopelintercept -0.0138/0.695 -0.0141/0.815 -0.0158/0.883 -0.0141/0.887
a Correlation coefficients in all cases were >0.97. The equation is of the form log h ’ = ~ m (% organic) + b, where m is the slope and b is the y intercept.
n
n
v
IN’ v
* f 0l 0 I
m
II
m
Figure 1. Structures of PCP and Identified cosynthetlcs: 1-(1pheny1cyclohexyl)piperidine (PCP, I); 1-[ 1-( 1, lf-blphenyl-4-yl)cyclohexyl]piperidlne (11); 1-[ 1-(phenylethyl)cyclohexyl]piperldine (111); ,4-phenylenedicyclohexylidine)bis[piperidine](IV). 1, lf-(l
Table I contains the slopes and intercepts of the least-squares equations determined for log k$ vs. solvent composition (determined using at least three different MeOH/H20 or CH&N/H20 solvent ratios) for the C1, Cg,c&and c8sodium sulfonates. For the 5.0 mM concentration of pairing ions, k’values were obtained for solvent compostions (MeOH/H20,MeCN/H20)varying between 30% and 90%. For the varying concentrations of C6SO3Na, k’ values were obtained at 2.5, 5.0, and 7.5 mM C6SO&3 at MeOH/H20 concentrationsof 60, 70, and 80%. Similar techniques were used for determining (Table 11, MeOH only) necessary for the selectivity ratio calculations.
RESULTS AND DISCUSSION In the crude chromatogram first obtained with the impure PCP synthetic mixture utilizing a moving phase of dilute acetic acid-heptanesulfonic acid in 80% MeOH:H,O on a (2-18 column, 11peaks were observed. Two of these, PCP (I) and 1-[1-(l,l’-biphenyl-4-yl)cyclohexyl] piperidine (11) were of sufficient intensity that, if kept on scale, the other minor component peaks became too small for accurate measurement. Further as the selectivity between I and I1 ( ( Y ~ I J I ) improved, comparable improvement was noted throughout the chromatogram and no k’inversions were observed such as those found by Sachok et al. (8) in their 2,6 disubstituted aniline study. In view of the parallel behavior of all peaks present relative to I and 11, the separation parameters studied herein were optimized by utilizing the large peaks observed for I and
11.
As shown in Table I, the absolute value of the slope (Islopel) of log k’1 vs. % MeOH increased as the chain length of the pairing ion increased, indicating that as the PCP-alkylsulfonate ion-pair became more hydrophobic, small changes in mobile phase strength produced increasingly large changes in retentions (k 1). Similarly, increasing the chain length of the pairing ion increased the value of the y intercept, corresponding to the log k $ value at 0% MeOH in the mobile phase. Although k 5 values became large in non-organic-containing mobile phases, the fact that the extrapolated intercepts increased systematically with increasing pairing ion chain length suggests that the value of the intercept can be used as an indication of the ability of a particular pairing agent to retain
Table 11. Least-Squares Equationsa for log k‘n vs. % MeOH in Mobile Phase column C-8 (2-18 CN pairing slope/ slope/ slope/ ion intercept intercept intercept CH,SO,Na C -0.0517/4.19 -0.0239/1.63 -0.0256/1.82 C,SO,Na C -0.0508/3.80 C -0.0509/3.96 -0.0251/1.75 C,SO,Na C,SO,Na C -0.0548/4.52 -0.0222/1.59 a Correlation coefficients in all cases were >0.97. The equation is of the form log h’11= m(%MeOH) t b where m is t h e slope and b is the y intercept. Compound I1 gave extremely unsymmetrical peaks on the C-8 column (see text). the analyte on a given column. Further, comparison of the equations obtained with individual columns using MeOH/H20 and a given pairing agent (Table I), shows that, for all pairing agents, both the Islope) and the intercept decreased in the order C-18 > C-8 > CN. This observation, in accord with the usual concept of reversed-phase chromatography, supports the proposition (22) that increasing the hydrophobicity of the column packing results in increased retention of the hydrophobic analyte for a given set of chromatographic conditions. Table I contains the results of a similar set of experiments performed using MeCN as the organic modifier in the mobile phase. For the C-18 and CN columns, increasing the length of the alkyl chain of the pairing ion resulted in an initial increase in Islopel of the equation relating log k’ to the % MeCN in the mobile phase followed by a sharp decrease. Conversely, the C-8 column is characterizedby an initial large Islopel at CH3S03Na,which decreased for C6S03Naand then increased for C ~ S O ~ Nremaining E~, essentially constant for C8S03Na. The change in y intercept (log k’,) parallels the slope change in each case. The effect of different columns on the same pairing agent is such that for the pairing agents C5S03Na,C6S03Na,and C8S03Na,both Islopel and intercept decrease in the order C-18 > C-8 > CN analogous to that obtained using MeOH as the organic modifier (see Table I). However, with CH3S03Naas the pairing agent, the order of decrease in Islopel and intercept is C-8 > C-18 > CN. Only the MeOH system is considered hereinafter, since: (1) as shown in Table I, MeOH provided a predictable and logical change of retention upon changing either the column or the pairing agent: (2) qualitatively, MeCN provided no unique selectively between PCP and PCP cosynthetics (vide infra); and (3) MeOH is considerablyless expensive than MeCN, an important consideration when running large numbers of samples. Thus, the selectivity between PCP and cosynthetics was examined as a function of column/pairing agent/ % MeOH-H20, for which a was calculated for I and I1 at MeOH/H20 concentrationsof 90,80,70,60, and 50% for each of the pairing agents. Plots of log vs. % MeOH in mobile phase were linear (correlation coefficients were better than
184
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982 ~
Table 111. Experimental and Predicteda Values for log k ' as ~ a Function of % MeOH and of Pairing Ion Chain Length on the C-18Column
\
k'I
% MeOH
exptl
predicted
log k'I exptl predicted
C,SO,Na
m
f
-m
2
45 50 60 65 80
0.638 0.428 0.0294 -0.143 -0.770
45 55 60 65 70
0.857 0.415 0.204 0.0414 -0.140
0.580 0.407 0.0508 -0.116 -0.639
4.34 2.68 1.07 0.72 0.1 7
3.80 2.55 1.14 0.77 0.23
C,SO,Na
1
c$':'CH3 O'
50
GO
7b
8b
Sb
160
55 60 75 80 90
% MeOH
0.9999) over the range of MeOH concentration examined for each pairing ion with the C-18 and with the CN columns (poor peak symmetry precluded further use of the C-8 column). Figure 2 illustrates the plots of log C U ~ vs. / I % MeOH obtained with the C-18 column and indicates that, for all MeOH concentrations, the CH3S03Napairing ion results in greater selectivity than the C5, c g , or C8 sulfonates. However, peak symmetry was poor with CH3S03Na,presumably because the shorter alkyl chain in the methylsulfonate anion is less able to shield the piperidyl nitrogen from active sites on the column. In addition, Figure 2 shows that, at high MeOH concentrations, the selectivity between I and I1 increases with increasing pairing ion chain length although at ca. 70% MeOH very little difference in selectivity was observed between C5, cg,and C8 sulfonates. However, as the concentration of MeOH was decreased below ca. 70%, the order of increasing selectivity was reversed so that the C5S03Nasystem showed the greatest selectivity and the C8S03Na system the least selectivity. The selectivity between I and I1 is obviously considerably greater with the C-18 column than with the CN column. Combination of the latter with a mobile phase of 60-70% MeOH in H 2 0 with either C6S03Na,CgSo3Na,or C8S03Na as the pairing ion could therefore be used for the routine analysis of PCP synthetic mixtures with fast analysis time and adequate separation of the cosynthetics. At higher MeOH concentrations, in contrast, early eluting components 111 and IV (Figure 6) are not well resolved from the large PCP peak while at lower MeOH concentrations retentions of the later eluting componentsare excessivelylong. The choice of pairing ion (C6S03Na)was made solely on the basis of ready availability since Figure 2 shows little difference in selectivity between C6, cg, and C8 sulfonates at 70% MeOH. The excellent linearity of log k $ vs. % MeOH and log k $ vs. chain length of pairing ion plots suggested the use of the "minimum alpha plots" (MAPS) technique of Sachok et al. (8) with chain length (nC) of the pairing ion (PI) and % MeOH as the independent variables. The k'values of I and I1 were fitted to an equation of the form log k'= a(% MeOH) + b(nC in PI) + c ( % MeOH)(nC in PI) d (1)
+
using the general linear models procedure of SAS (13). That
7.19 2.60 1.60 1.10 0.71
6.81 2.75 1.75 1.11 0.71
C, S0,Na
'
Flgure 2. Plot of log a,,,, as a functlon of % MeOH for various pairlng ions on the C-18 (-) and CN (- -) columns.
0.833 0.439 0.243 0.0462 -0.1 50
0.927 0.543 -0.125 -0.387 -0.796
0.853 0.612 -0.112 -0.353 -0.835
8.45 3.49 0.75 0.41 0.16
7.12 4.09 0.77 0.44 0.15
a Predicted values obtained from eq 1;a = -0.0125, b = 0.453, c = -0.00447, d = -0.116; correlation coefficient = 0.995.
2
2.06
0 03
Figure 3. Plot of log k', (z axis) as a function of % MeOH (x axis) and chaln length of pairlng ion (yaxis) on the C-18 column.
eq 1 is a legitimate model is demonstrated in Table I11 which lists both the experimental and predicted values of log kl, and k'with the C-18 column, along with the derived parameters, a, b, c, and d. The data show that, as the length of the pairing ion decreases, the agreement between predicted and experimental k' also decreases. This beginning nonlinearity of eq 1at CsSO3Napredicts that the pairing ion CH3S03Nawould not, as observed, follow the relationship defined. Indeed, a plot of log k'vs. nC in PI at constant % MeOH showed that CH3S03Nadata deviated appreciably from the line defined by Ck8S03Na,conceivably due to the increasing interaction of the less protected piperidyl nitrogen with active sites on the column which also results in the poor peak symmetry previously described. Figure 3 shows a three-dimensional plot of the plane generated (using the SAS/graph procedure) (14) by solving eq
ANALYTICAL CHEMISTRY, V M . 54. NO. 2. FEBRUARY 1982 185
Figure 5. a,,,, (zaxis) as a function of % N O H (x axis) and concentration of hexanesulfonate (mM) (v axis). F C l v 4. a,,,, (zaxis) as a functionof % MeOH (x axis)and pMng ion chain length (v axis) on the G I 8 column. a values greater lhan 3.5 are set equal to 3.5. Axes are tilted and rotated 45’.
1for log k; (with appropriate a, b, c, and d parameters) at MeOH percentages of 3040% a t 1% intervals and nC in PI from 5 to 8 a t 0.2 carbon intervals. It should be noted that, although pairing ions cannot actually posseas fractional chain lengths, the 0.2 carbon interval chosen for the pairing ion axis can he achieved by “pairing ion averaging”. For example, it has been demonstrated that a 5050 mixture of C7S03Naand C,S03Na yields the equivalent of CsS03Na (15). Similar linearity was observed for log kh vs. % MeOH and chain length of the pairing ion reagents with the C-18 column. The a, b, c, and d parameters of eq 1for compound I1 were a = 4.0668,b = -0.118, c = 0.00304, and d = 4.47, and the correlation coefficient was 0.94. Figure 4 shows the three-dimensional plot of selectivity (a) of I and II as a function both of % MeOH and of pairing ion chain length with the C-18 column. The plot was generated using the SAS/graph procedure, and solving lot eq 1for compd I1 an’1 = lOteq 1 for compd I
for MeOH percentages of 3(t90% at 2% intervals and pairing ion chain length 5 to 8 at 0.1 i n t e ~ & . For the sake of clarity, values of a greater than 3.5 were set equal to 3.5. The valley corresponds to the worst separation conditions for I and 11. The area centered at ca. 65% MeOH and at the pairing ion chain length of about 6.5 corresponds very closely to the optimum conditions predicted from inspection of the twodimensional plot of Figure 2. Thus, in this instance, it is possible to optimize in “three dimensions” using a two-dimensional plot, thereby avoiding use of (frequently unavailable) software and hardware necessary for three-dimensional plotting. I t should be noted, however, that this approach will not simultaneously optimize more than two a m ponents in the mixture unless all components are similarly affected by changes in separation parametem as was observed with the PCP synthetic mixtures. The area ’behind” the valley in Figure 4 also yields high values of a,hut separation times are here prohibitively long. Since the concentration of the ion-pairing agent can have a profound effect on selectivity (S),we also investigated the variation of a as a function of % MeOH and pairing ion
A ‘
5
’
Fbwe 6. PCP synthetic mixture at 0.005 AUFS. Condnions: pBoKlapk G I 8 column: 7 0 3 0 MeOH:H,O 5 mM C,SO,Na: pH 3.5: flow rate 2.5 mLlmin. Peak identification I-IV as in text. others unknown. concentrations. Figure 5 shows the three-dimensional plot of a as a function of % MeOH and CsS03Na concentration generated in a fashion analogous to Figure 4. The separation of I and I1 is essentially independent of CsSO3Na concentration in the range of % MeOH and C&OsNa concentrations examined (6C-SOW and 2.5-7.5 mM, respectively),despite the fact that log k‘of both I and I1 was found to increase linearly with increasing C&03Na concentration. However, no change in 01 occurred (increases of k; and kh nearly parallel) in contrast to the findings of Sachok et al. (8)where selectivity was a function of pairing ion concentration. Figure 6 illustrates a chromatogram of the crude base fraction of the described PCP synthesis with a C-18 column and a mobile phase of 7030 MeOH/H,O, 5 mM C&03Na AU four identified compounds are separated along with seven unidentified components and the entire run required leas than 8 min. We have since examined 20 different PCP samples synthesized in this laboratory and 4 samples seized from illicit manufacturers or distributors. No two samples gave identical
188
Anal. Chem. 1982, 54, 186-189
chromatograms (relative peak intensities), suggesting that “fingerprint” types of chromatograms could be of some forensic utility (16). In summary, the chromatographic behavior of PCP has been determined on C-18, (2-8, and CN columns using methyl-, n-pentyl-, n-hexyl-, and n-octylsulfonates both with aqueous MeOH and with MeCN mobile phases. The selectivity between PCP (I) and the cosynthetic I1 has been determined, and a system for routine analysis of PCP cosyntheticmixtures has been developed. The applicability of the system has been demonstrated on synthetic PCP mixtures.
Gloor, R.; Johnson, E. L. J . Chromatogr. Sci. 1977, 15, 413-423. Sachok, 6.; Stranahan, J. J.; Demlng, S. N. Anal. Chem. 1981, 53, 70-74. Lurle, I . S.; Demchuck, S. M. J . Liq. Chromatogr. 1981, 4 , 337-355 and 357-374. Jones, LIA.; Beaver, R. W.; Schmoeger, T. L. J. Org. Chem. 1981, 46, 3330-3333. Snyder, L. R.; Kirkland, J. J. “Introduction to Modern Liquid Chromatography”, 2nd ed.; Wlley: New York, 1979. Horvath, C.; Melander, W. J. Chromatogr. Sci. 1977, 75, 393-404. Helwlg, J. T., Council, K. A., Eds. “SAS User’s Gulde-1979 Edition”; SAS Institute: Raleigh, NC, 1979. Councll, K. A., Helwig, J. T., Eds. “SASIGraph User’s Guide-1961 Editlon”; SAS Institute: Cary, NC, 1961. Waters Assoclates Llquid Chromatography Training Manual, p LS-25. Wheals, B. B.; Smlth, R. N. J. Chromatogr. 1975, 705, 396-400.
LITERATURE CITED Kalir, A.; Edery, H.; Pelah, Z.;Balderman, D.; Porath, G. J . Med. Chem. 1969, 12, 473-477. Shulgln, A. T.; MacLean, D. E. Clln. Toxlcol. 1978, 9 , 553. Baker, J. K.; Skelton, R. E.; Ma, C.-Y. J . Chfomatogf. 1970, 168, 417-427. Jaln, N. C.; Leung, W. J.; Budd, R. D.; Sneath, T. C. J . ChfOm8togf. 1975, 775, 519-526. Lin, D. C. K.; Fentiman, A. F., Jr.; Foltz, R. L.; Forney, R. D., Jr.; Sunshlne, I. Blomed. Mass Spectrom. 1975, 1,206-214. Pitts, F. N., Jr.; Yago, L. S.; Anillne, 0.; Pitts, A. F. J . Chromatogr. 1980, 193, 157-159.
RECEIVED for review July 27,1981. Accepted October 26,1981. The authors are grateful for the generous financial support of the North Carolina Department of Crime Control and Public Safety, Division of Crime Control, and the Biomedical Research Support Grant No. RR07071. Presented in part at the combined Southeast/Southwest Regional Meeting of the American Society, New Orleans, LA, 1980.
Determination of Tetrahydroisoquinolines by Reversed-Phase Liquid Chromatography with Gradient Elution and Amperometric Detection R. L. St. Clalre 111, G. A. S. Ansarl, and Creed W. Abell” Department of Human Biological Chemistry and Genetics, Division of Biochemistry, Universiv of Texas Medical Branch, Galveston. Texas 77550
Reversed-phase chromatography with a itnear gradient of 2-propanoi against a constant concentration of aqueous acetic acid is used to resolve 3,4-dihydroxybenzylamine, dopamine, and six different 6,7-dihydroxytetrahydrolsoqulnollnes. We propose that acetic acid functlons not only in ion suppression of these compounds’ acidic groups by iowerlng pH but as a source of anions (acetate) which shleid the amine function from column silanols such that capaclty factors ( k ’ ) and selectivity (a)can be manlpulated for optimum resolution. Simultaneous determlnatlon of 1.25 pmoi of each compound under gradlent elution wlth a relative standard devlatlon of 20% (n = 4) Is achleved with amperometrlc detection. The applications of isocratlc elution and hydrodynamic coulometry are also examined.
Tetrahydroisoquinolines, a class of isoquinoline alkaloids, are synthesized from the bimolecular condensation of biogenic amines, such as dopamine, with aldehydes or a-keto acids (1). The presence of these compounds in the normal human population and in disorders such as Parkinson’s disease, phenylketonuria, and alcoholism is currently being investigated (for review, see ref 2). Unlike the determination of tetrahydroisoquinolinesby gas chromatography with electron capture and combined gas chromatography/mass spectrometry (3-5)) liquid chromatography with amperometric detection does not require volatile derivatives, and the compound is essentially preserved for 0003-2700/62/0354-0186$01.25/0
further identification following detection. Two types of liquid chromatography are used: cation exchange and reversed phase. Cation-exchange chromatography has been the method of choice, primarily because tetrahydroisoquinolinescan carry a charged amine at low pH (6),but resolution between tetrahydroisoquinolines decreases significantly as they become more hydrophobic. Reversed-phase chromatography, however, has the potential for improved sensitivity and resolution of most tetrahydroisoquinolines (7). Reversed-phase chromatography of some 6,7-dihydroxytetrahydroisoquinolineshas been reported (4,8), but insufficient chromatographic data have been published to allow critical evaluation of this method’s versatility or efficiency. Published reports on the amperometric detection of these compounds have not provided data on the linearity and precision of the detection process. The objectives of this study were (1) provide a versatile reversed-phase separation technique for 6,7-dihydroxytetrahydroisoquinolines and (2) document the sensitivity of amperometric detection as applied to this chromatographic system. Six different 6,7-dihydroxytetrahydroisoquinolines, dopamine, and 3,4-dihydroxybenzylamine(used as an internal standard in the analysis of biological samples) were used to illustrate these objectives.
EXPERIMENTAL SECTION Apparatus. Solvent delivery was performed by using dual Beckman 110A single-piston pumps with pulse dampers. Chromatography was achieved on a 5-pm Ultrasil-ODS (4.6 X 250 mm) reversed-phase column in which temperature was maintained by a Brinkman K-2/R water bath and an Alltech 30-cm water jacket. 0 1962 Amerlcen Chemical Soclety
