Anal. Chem. 1907, 59, 266-270
266
catecholamines. While this is higher than the limits of detection reported for amperometry, the sensitivities are comparable to other HPLC-RSCSV techniques (1,3). Figure 8 shows the reductive RSCSVs of trace amounts of p-benzoquinone and 9,lO-phenanthrenequinone.It is of interest that picomole levels can be measured even when the CV peaks are highly cathodic. Registry No. p-Benzoquinone, 106-51-4; 9,lOphenanthrenequinone, 84-11-7;g,lO-anthraquinone, 84-65-1; 1nitroanthraquinone, 82-34-8;dopamine, 51-61-6; noradrenaline, 51-41-2;andrenaline, 51-43-4;estone, 53-16-7; estradiol, 50-28-2; estriol, 50-27-1.
(a!
LITERATURE CITED 151 L
+
05
-05
-1 5 E I V vs Ag/Ag+
Flgure 8. RSCSV of trace amounts of quinones: (a) 3.1 ng of 9,lOphenanthrenequinone and (b) 3.2 ng of p-benzoquinone. Eluent was 60140 ethanollhexane, 0.1 M tetrabutylammonium fluoroborate, and 0.1 M acetic acid. Flow rate was 1.O ml min-’ and scan rate was 1.25 v s-’.
of voltammetric peak currents (I,) and peak potential (E,) values for the various test compounds. The LC-RSCSV technique was linear for amounts varying from picomole to nanomole levels. The detection limit was found to be of the order of picomole levels for the oxy and nitro derivatives of PAHs and nanomole levels for the oestrogen steroids and
(1) Samuelsson, R.; O’Dea, J.; Osteryoung, J. Anal. Chem. 1980, 5 2 , 2215. (2) Thorgeson, N.; Janata, J.; Ruzicka. J. Anal. Chem. 1983, 55, 1986. (3) Caudill, W. L.; Ewing, A. G.; Jones, S.; Wlghtman R. M. Anal. Chem. 1903, 5 3 , 1877. (4) Hughes, S . ; Johnson, D. C. Anal. Chlm. Acta 1981, 132, 11. (5) Wang, J.; Ouziel, E.; Yarnltsky, Ch.; Ariel, M. Anal. Chim. Acta 1978, 702, 99. (6) Ploegmakers, H. H. J. L.; Mertens. M. J. M.; Van Oort, W. J. Anal. Chim. Acta 1985, 174, 71. (7) White, J. G.; St. Claire, R. L., 111: Jorgenson, J. W. Anal. Chem. 1988, 5 8 , 293. (8) Scanlon, J. J.; Flaquer, P. A,; Robinson, G. W.; O’Brien, G. E.; Sturrock, P. E. Anal. Chim, Acta 1984, 158, 169. (9) Wang, J.; Dewald, H. b. Anal. Chim. Acta 1983, 753, 325. (10) Trojanek, A.; De Jong, H. Anal. Chim. Acta 1982, 747, 115. ( 1 1 ) Kissinger, P. T. Anal. Chem. 1977, 49, 447A. (12) Miaw, L. H. L.; Perone, S. P. Anal. Chem. 1979, 51, 81 1. (13) Gunasingham, H.; Tdy, B. T.; Ang, K. P. Anal. Chem. 1984, 5 6 , 2422. (14) Meyer, G.; Nadjo, L.; Saveant, J. M. J . flectroanal. Chem. 1981, 179,417. (15) Gunasingham, H.; Tay, B. T.: Ang, K. P. J . Chromatogr. 1985, 341, 271. (16) Gunasingham, H.; Fleet B. Anal. Chem. 1983, 55, 1409. (17) Gunasingham, H.; Tay, B. T.; Ang, K. P. Anal. Chim. Acta 1985, 776, 143.
RECEIVED for review April 22, 1986. Accepted September 16, 1986.
Determination of Ivermectin by Fluorescence Derivatization John D. Stong Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
Reaction of 5-keto derivathres of various 22,23-dlhydroavermectlns with ammonium acetate In ethanol produces an Intensely fluorescent product (A, 445 nm with A,, 361 nm) Identifled by ’H NMR as the 7-deoxy-2,5,6,7-tetrahydro22,23-dlhydroavermectlns.Both the oxidation of 22,234hydroavemctln B,, (H2B1,) to the correspondhg 5ketone by MnO, and its subsequent conversion to the fluorescent derivative are reproducible and Hnear over the parent avermectin concentration range of 0.01-10 jig/mL. The percent devlatlon from the regresslon Hnes was 3% for the range 1-10 jig of H,Bl,/mL, 8 % for the range 0.1-1 jig of H2B1J mL, and 12% for the range 0.01-0.1 jig of H,B,,/mL. A mechanism for the reactlon Is proposed and several factors affectlng the anaiytlcal reaction are examined.
The avermectins are a group of pentacyclic 16-membered lactones related to the milbemycins (1, 2). Of particular interest is ivermectin, a mixture of >80% 22,23-dihydroav-
ermectin B,, (H2Bla, 1) and The tubes were capped and shaken for 30 min, after which time they were centrifuged and 15 mL of the supernatant solution was filtered through a Gelman 0.45-pm Acrodisc. A 10.0-mL aliquot of this filtered solution was transferred to a 15-mL conical centrifuge tube and evaporated under a stream of pure N2 at 60 "C. After the sample cooled, 0.5 mL of EtOH saturated with ammonium acetate was added, the tubes were capped and vigorously agitated for 30 s, and the contents were transferred to 1-mL Pierce Reacti-vials fitted with Teflonlined caps. The vials were placed in a Pierce Reacti-block at 75 "C for 45 min. Chromatography performed on these solutions containing the fluorescent derivative 8 was the same as that described for the kinetics experiments.
+
RESULTS AND DISCUSSION Structural Assignment of the Fluorescent Derivative. When 5 or 6 is dissolved in ethanol containing ammonium acetate, an intense blue-white fluorescence gradually develops. The electronic spectrum is dominated by an intense absorption at 269 nm, with a broad, less intense (by about 314) band centered a t about 345 nm with a maximum at 361 nm. The fluorescence emission maximum at 445 nm occurs with excitation a t 361 nm. Removal of the oleandrosyl-oleandrosyloxy side chain from C13 of the macrocycle results in a somewhat simplified 'H NMR spectrum, particularly in the region pertaining to most of the C2-C7 ring system ( I ) . The spectra obtained from H2Bla-5-oneaglycon, the fluorescent derivative, and this derivative shaken with D20 are presented in Figure 2. Pertinent assignments are listed in Table I and are based upon changes noted in the spectrum of H2B,,-aglycon upon oxidation, together with assignments published for avermectin A, aglycon ( I ) , avermectin Bla bis(sily1 ether) ( I I ) , and avermectin Bza-5-one (12). The NMR spectra reveal structural changes only in the C2
