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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
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982 187
HO
SAL NCCA
R = H R =CH, R’COOH R =CH2 C6H4 OH(p)
HO
DHSA R”CH2-NH2 DA R=CH,-CH,-NH,
MNLCA R’COOH
R ‘CH2 C6H3:0CH3(m1 OHipl .OHlml R = H R = CH2 C6H3. THP OHipl DNLCA R COOH R = CH2 C6H5 HG R = H R = CH2 C,H, OHipl
Flgure 1. Cataecholamlnes and dopaminederived 6,7dlhydroxytetrahydrolsoquinollnes.
Amperometric detection was performed with a Bioanalytical Systems TL-5 glassy carbon transducer and LC-3 amperometric controller. An Altex C-R1A data processor integrated and printed the data. Reagents. Alcohols and water used in the mobile phase were liquid chromatographygrade (Burdick and Jackson). Additional mobile phase constituents included glacial acetic acid (Reagent ACS) and disodium ethylenediaminetetraacetate(CertifiedACS), both from Fisher Scientific Co., and n-nonylamine (99%) from Aldrich Chemical Co. Dopamine-hydrochloride (99%) (DA) and 3,4-dihydroxybenzylamine-hydrobromide (98%) (DHBA) were also purchased from Aldrich. (f)-Salsolinol-hydrobromide (SAL) and (f)-tetrahydropapaveroline-hydrobromide (THP) were provided courtesy of Smith, Kline, and French Laboratory, Philadelphia, PA. (f)-Norcoclaurinecarboxylic acid-water (NCCA) and (f)-higenamine-hydrobromide (HG) were kindly provided by Robert V. Smith of the Drug Dynamics Institute, University of Texas, Austin, TX. (f)-3’-Methoxynorlaudanosolinecarboxylicacid (MNLCA) was provided courtesy of Arnold Brossi of the National Institute of Arthritis, Bethesda, MD. (~)-3’,4’-Deoxynorlaudanosolinecarboxylic acid-hydrochloride (DNLCA) was prepared in this laboratory according to published procedures (8). For structures refer to Figure 1. Procedure. DHBA (1.10 mg), DA (0.948 mg), SAL (1.30 mg), NCCA (1.67 mg), MNLCA (1.73 mg), THP (1.84 mg), DNLCA (1.68 mg), and HG (1.76 mg) were individually and as a mixture added to 2 mL of 1.0 M HCI. Addition of 8 mL of H20 to this solution followed by rapid momentary heating to 70 O C , with mixing, dissolved all compounds with no detected loss of any standard. This solution was diluted to a final concentration of 1 X lob M for each compound. This stock solution could be stored safely at -30 “C for at least 1 month. The stock solution was further diluted with mobile phase prior to use. All stock solutions were processed prior to injection with a Bioanalytical Systems’ MF-1 (0.2-pm) centrifugal microfilter. In addition to the specific conditions listed with Figures 2-6, the following general procedures applied to all experiments: (1) 20 pL of an equimolar mixture of all eight compounds was injeded with a Monoject insulin syringe to eliminate amperometrically active metal ions which were formed with a conventional syringe, (2) chromatographywas conducted at a flow rate of 1.5 mL/min with a column temperature of 26 O C , and (3) except as noted in Figure 5, an applied potential of +725 mV vs. Ag/AgCl was used. All mobile phases were fiitered with Millipore HA (0.45pm) fiiters.
RESULTS AND DISCUSSION Chromatography, Our approach to the reversed-phase separation and amperometric detection of tetrahydroisoquinolines was to optimize chromatography with a mobile phase that contained an electrolyte, had a high dielectric constant, and was electrochemically inert at the applied potential. To achieve this, we used a mobile phase containing acetic acid and 2-propanol delivered in a linear gradient. Because these amines differ significantly in polarity, gradient elution was employed. 2-Propanol was used, as opposed to methyl or ethyl alcohol, because for any given separation time it reduced the dielectric constant of the mobile phase the least. This occurred because the greatly reduced gradient range (v/v) required with 2-propanol compensated for the fact that 2-propanol has the lowest dielectric constant. This di-
03
10
15
20
25
30
% Acetic Acid (v/vl
Figure 2. Effect of percent acetic acid (v/v) on k’ under gradient elution. Mobile phase: A = (0.3-3.0%) acetic acid (v/v) and B = (0.3-3.0%) acetic acid (v/v) with 10% 2-propanol (v/v). Solvent delivery: starting at 0.0 mln from 10 to 75% B over 10 min (linear). Sample injected: mixture wlth 200 pmol of each compound. Sensitivity: 100 nA full scale.
electric effect was important because chromatography and amperometric sensitivity would be affected if the alcohol concentration was high enough to increase the pKs of acetic acid such that there would be a significant increase in pH and decrease in ionic strength. Optimum gradients are described in the appropriate figure captions. Catecholamines and 6,7-dihydroxytetrahydroisoquinolines, which have phenolic and carboxyl groups, undergo not only regular reversed-phase equilibria based on polarity but also secondary chemical equilibria based on ionizations of the molecule. By lowering the pH of the mobile phase, ion suppression increases the k ’ of acidic compounds on reversedphase and improves peak symmetry. For the weakly acidic groups common to our compounds, a mobile phase pH of 2-3 is sufficient to shift equilibrium toward the un-ionized form (9). Acetic acid (pK, = 4.76) was used for this purpose. The effect of percent acetic acid (v/v) on k’under gradient elution is shown in Figure 2. From 0.3 to 3.0% acetic acid (v/v), the k’ for all compounds decreased, while selectivity (CY)between compounds either changed or remained constant. For each percent acetic acid (v/v) tested, the mobile phase pH did not change during the 2-propanol gradient. Mobile phase pH decreased from 3.3 to 2.9 as acetic acid concentration was increased from 0.3 to 3% (v/v). For each percent acetic acid (v/v) examined, solvents (A) and (B) (Figure 2) maintained a constant acetic acid concentration during the gradient, and because a pH change was not detected during the gradient, the ionic strength was assumed to remain constant as well. Figure 3 illustrates the extent to which resolution (based on capacity factors and selectivity) was affected under the conditions of Figure 2. The effect of acetic acid on k’and CY under isocratic elution, using acetic acid and 2-propanol concentrations within the range used in Figure 2, is illustrated in Figure 4. The k’for all compounds at any percent 2-propanol (v/v) was lower with 3% acetic acid (v/v) (A) than 1%acetic acid (v/v) (B), and the reversal in elution order between DNLCA and HG depended solely on the percent acetic acid. The effects of pH and ionic strength on k’ and 01 were examined. Adjustments of the pH of the 0.3% acetic acid (v/v) mobile phase (pH 3.3) to the pH of the 3.0% acetic acid (v/v) mobile phase (pH 2.9) and pH adjustment in the reverse direction did not alter k’or CY, as shown in Figures 2-4. The ionic strength effect of acetic acid was determined from the pattern in Figure 4 at 5% 2-propanol (v/v). The approximate acetate concentrations of a 1% and 3% acetic acid solution (v/v) were determined. When sufficient NaCl was added to 1%acetic acid (v/v) to yield a mobile phase with approxi-
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A
B
--
I
0
4
C
0 4
8 1216202428
8 1216
0 4 8 1216
Minutes
Figure 3. Elution profile of 200 pmol of each compound under a constant gradlent of 2-propanol with 0.3% (A), 1.5% (B), and 3.0% (C) acetic acid (v/v). Conditions identical with those listed for Figure 2.
% 2-Propanol (v/v)
* DHSA
A DA
I
2.5
I
I
I
I
I
I
I
50
I
I
I
75
% 2-Propanol (v/v)
Figure 4. Effect of 3 % (A) and 1 % (8) acetic acid (v/v) on k’ under isocratlc elution. Mobile phase: A = (1% and 3%)acetic acid (v/v), and B = (1% and 3%)acetic acid (v/v) with 10% 2-propanol (v/v). Solvent delivery: isocratlc (25-75 % B). Sample injected and sensitivity: see Figure 2. mately the same anion concentration as a 3% acetic acid solution, k and a changed as predicted in Figure 4 for a 3% acetic acid (v/v) solution. In fact when two saline solutions adjusted to pH 3.0 and containing sufficient NaCl to approximate the anion concentration of a 1% and 3 % acetic acid solution were compared, k’ and a differed as predicted by Figure 4. Buffers were not used in this analysis because of their unavoidable contribution to ionic strength. Two different observations illustrated why increased ionic strength affected the k’and a of these compounds. First, the effects of acetic acid on k’and a have been observed with three different Ultrasil columns. With each new column, the optimum acetic acid concentration was 1.25% acetic acid (v/v) (refer to Figure 2), but after a period of several months, k’ increased and loss of resolution due to decreased a occurred. Only increasing the acetic acid to as much as 2.0% (v/v) would restore original k and a values. Under normal use, a likely change in column integrity with time is an increase in exposed
silanols. The second observation concerned the addition of n-nonylamine to the mobile phase which reacts with exposed column silanol groups and is recommended for the reversedphase chromatography of basic compounds (IO). Under the conditions of Figure 3B, with 1 X 10“ M n-nonylaminein the mobile phase, the chromatography changed from (B) to (C) (Figure 3) after 2 days of using the same mobile phase. This situation was reversed after the amine was removed from the mobile phase. This indicates that n-nonylamine: (1) accumulated in the column and (2) changed k’and a values as if the acetic acid concentration had been increased. We propose that factors which prevented the association of the charged amine common to these compounds with the column silanols changed k’and a in the manner described, and acetic acid as a source of anions (acetate) shielded the charged amine group from reactive silanols. Most importantly, if the ionic strength (acetate) was such that the a of THP/ DNLCA, THP/MNLCA, or HG/DNLCA was about unity for a particular column (refer to Figure 2), no gradient of 2propanol would have achieved resolution. This type of ion pairing has been demonstrated in reversed-phase chromatography of peptides, where the presence of phosphate is proposed to shield protonated amino groups, thus improving efficiency (11). Column efficiency was determined by using THP under the conditions listed for Figure 3B. For the new column used in Figures 2 and 3, the height equivalent to a theoretical plate (H) was 0.03 mm. Four months later when amperometric sensitivity was determined (Figure 6), H was increased 50%. Our chromatographic technique could be extended to the resolution of other related compounds. To modify the conditions used in Figure 3B to accommodate additional compounds with intermediate elution volumes, we could reduce the gradient delivery of 2-propanol, thereby substantially increasing the a of NCCA/SAL, HG/DNLCA, and THP/ MNLCA. Elevated temperatures and increases in 2-propanol concentrations to about 15% (v/v) could be employed for resolution of tetrahydroisoquinolines with substantial 0methylation and of more nonpolar isoquinolines such as tetrahydroberberines. From the opposite perspective, Figure 4 illustrates that when the separation requirement is relaxed, isocratic elution could be used. Amperometric Detection. Once the general requirements for separation and detection of these compounds were met, it became necessary to resolve two specific problems that affected the lower limits of amperometric sensitivity. First, with amperometric sensitivity at 5 nA full scale, background current dropped significantly during the gradient employed in Figures 2 and 3. Therefore, the gradient slope was reduced from 1.0 to 7.5% 2-propanol (v/v) over 10 min to the same range over 20 min. Second, a metal ion peak generated by the injection process masked DHBA and DA at 5 nA full scale. Maintaining a constant level of 2 X M disodium EDTA throughout the gradient solved this problem. Higher levels of EDTA (2 X M, for example) aggravated the shift in background current normally present during the gradient and changed k’ and a values. From the hydrodynamic coulogram (Figure 5), optimum sensitivity was indicated with an applied potential of +650 to 750 mV vs. Ag/AgCl. An attempt to extend the applied potential beyond +800 mV to complete oxidation of MNLCA and to induce oxidation of the phenolic groups of HG and NCCA was abandoned due to very high background current. Figure 5 shows that at “maximum oxidation”, THP with ita two catechol groups generated about twice as many coulombs as SAL with its single catechol group. HG with its single catechol group, however, generated significantly fewer coulombs than DNLCA, which also has a single catechol function.
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800
0.$nA
A
Figure 8. Reversed-phase separation of a mixture containing 1.25 (A) 10 (B) pmol of each compound. Mobile phase and solvent delivery:
and
- -
.....
.
Applied Potential ( V I
Figure 5. Hydrodynamic coulogram of a mixture containing 100 pmol of each compound. Mobile phase: A = 1.25 % acetic acid (v/v), with 0.2 mM disodium EDTA and B = 1.25% acetic acid (v/v) with 0.2 mM disodium EDTA, and 10 % 2-propanol (v/v). Solvent delivery: starting at 0.0 min from 10 to 7 5 % B over 20 min (linear). Sensitivity: 50 nA full scale.
HG appeared to exhibit a lower diffusion coefficient than DNLCA in this mass transport limited process. Under different circumstances, however, large differences in coulombs per mole injected between experimental and expected values for a given type of molecule could indicate an impurity in the compound. Because THP had substantial coulombs per mole injected even at +450 mV, it might be desirable to study isoquinolines with multiple catchol functions using lower applied voltages because detection limits might be improved as electrochemical selectivity would be increased and background current decreased. Response factors (pC/pmol injected) were determined by using a detector sensitivity of 5 nA full scale at +725 mV vs. Ag/AgCl. A linear response for all compounds was demonstrated from 1.25 to 10.0 pmol of compound injected. The actual chromatograms corresponding to the low (A) and high (B)point of this range are shown in Figure 6. When detector sensitivity was changed to 100 nA full scale, linearity was shown to extend to 200 pmol injected for each compound. Also, all compounds demonstrated linear performance (d(retention time)/d(amount injected) = 0) from 1.25 to 200 pmol injected. Precision in the determination of peak areas for each compound throughout the range of 1.25-10 pmol injected was measured by relative standard deviation ( s / X ) . In mixtures containing 1.25 pmol of each standard with n = 4, s / X = 0.11-0.27. For 2.5 pmol, s / X = 0.034.11, and this degree of precision remained constant through 10 pmol injected. Such changes in precision throughout the concentration range were due to the integrator, not the amperometric cell. The integration technique employed in Figure 6 used automatic drift
correction and a slope change sensitivity set just low enough to recognize a peak height of two times background current. Minimum area recognition was manipulated to just below the smallest important peak so that the background current of 63 pA was “ignored”. If simultaneousdetection of a variety of compounds (Figure 6) is not required, a change to an isocratic mode would: (1) reduce background current oscillations, since only one pump would be pulsating and (2) reduce the background current’s downward shift, due to the gradient itself. These two factors would allow detector sensitivity to be expanded to at least 2 nA full scale with no compromise in integration precision. ACKNOWLEDGMENT We express our grateful appreciation to Glen Scratchley for helpful comments and encouragement and to Peter Kissinger for his guidelines on reporting performance figures for amperometric detection. LITERATURE CITED (1) Shamma, M. “The Isoquinoline Alkaloids: Chemistry and Parmacoiogy”; Academic Press: New York, 1972. (2) Collins, M. A. Adv. Exp. Med. Biol. 1980, 126, 87-102. (3) Collins, M. A.; Nijm, W. P.; Borge, G. F.; Teas, G.; Goldfarb, C. Science 1979, 206, 1184-1186. (4) Orlgitano, T. C.; Collins, M. A. Life Sci. 1980, 26, 2061-2065. (5) Lasala, J. M.; Coscia, C. J. Science 1979, 203, 283-284. (6) McMurtrey, K. D.; Cashaw, J. L.; Davis, V. E. J . Liq. Chromatogr. 1980, 3, 663-679. (7) Riggln, R. M.; Kissinger, P. T. Anal. Chem. 1977, 4 9 , 530-533. (8) Hudlicky, T.; Kutchan, T. M.; Shen, G.; Sutiiff, V. E.; Coscia, C. J. J . Org. Chem. 1901, 4 6 , 1736-1741. (9) Majors, R. E. “High-Performance Llquid Chromatography, Advances and Perspectives”; Horvith, C., Ed.; Academlc Press: New York, 1980; Vol. 1, p 95. ( I O ) Cooke, N. H. C.; Olsen, K. J . Chromatogr. Sci. 1980, IS, 512-524. (11) Hancock, W. S.; Bishop, C. A.; Prestidge, R. L.; Hardlng, D. R. K.; Hearn, M. T. W. J . Chromogr. 1970, 153, 391-398.
RECEIVED for review August 10,1981. Accepted November 3,1981. This work was generously supported by the Cullen Foundation and the Multidisciplinary Research Program on Schizophrenia.
