Anal. Chem. 1994,66, 4079-4084
Improved Peroxyoxalate Chemiluminescence-Based Determinations by Use of Continuous Reagent Addition To Remove Background Emission Juana Cepas, Manuel Silva, and Dolores P6rez-Bendlto' Department of Analytical Chemistry, Faculty of Sciences, University of Cbrdoba, E- 14004 Cbrdoba, Spain
The continuous addition of reagent technique was used as an effective means for suppressing background emission in peroxyoxalate chemiluminescencereactions. For this purpose, the bis(2,4,6-trichlorophenyl) oxalate/hydrogen peroxide system and phenothiazine derivatives such as acepromazine, propiopromazine, thioridazine, promazine, trimeprazine, methotrimeprazine, and chlorpromazine were chosen. The influence of environmental factors such as pH, water content, reagent concentration,and addition rate on the maximum reaction rate was assessed in terms of background emission. The resulting method provides a linear response to phenothiazinederivatives over a range of 3 orders of magnitude with relative standard deviation of 1-3.7% and detection limits in the picomole range for some phenothiazine derivatives, in addition to a high selectivity. The proposed method for acepromazine and propiopromazine exhibits the best analytical features and compares favorably with existing alternatives in terms of sensitivity, precision, and sampling frequency. The determination of acepromazine in horse plasma was selected to validate the proposed method for analysis of real samples.
-
Peroxyoxalate chemiluminescence is a useful tool for the sensitive and some times selective determination of fluorescent analytes or fluorescent-labeled analytes in a great variety of samples.' Some examples include polycyclic aromatic hydrocarbons (PAHs) in coal tar extracts,2amino-PAHs in oils3 and biomass fuels,4dansyl-amphetamine-related compounds in urine,5 estradiol in serum,6 carboxylic acid in oxidized petroleum-based lubricating oils? etc. In the process, the fluorescent species is chemically excited by energy transfer from a intermediate formed by oxidation of an oxalate derivative [e.g., bis(2,4,6-trichlorophenyl)oxalate reacts with hydrogen peroxide for form an energetic intermediate, 1,2dioxetanedione, which transfers its energy to a fluorophor] . This form of fluorophor detection has two major advantages over classical spectrofluorometry, namely, (a) improved selectivity resulting from the fact that not all fluorophors are equal efficient as energy acceptors and (b) enhanced detection arising from suppression of source instability and source scatter in fluorescence detection. On the other hand, one serious (1) Lewis, S. W.; Worsfold, P. J. Anal. Proc. 1992, 29, 10-11. (2) Sigvardson, K. W.; Birks, J. W. Anal. Cfiem. 1983, 55, 432-435. (3) Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Cfiem.1984,56,10961102. (4) Yan, B.; Lewis, S.W.; Worsfold, P. J.; Lancaster, J. S.;Gachanja, A. Anal. Cfiim. Acra 1991, 250, 145-155. ( 5 ) Hayakawa, K.; Hasagawa, K.; Imaizumi, N.; Wong, 0. S.;Miyazaki, M. J . Cfiromalogr.1989, 464, 343-352. (6) Nozaki, 0.; Ohba, Y.; Imai, K. Anal. Cfiim. Acta 1988, 205, 255-260. 0003-2700/94/0366-4079$04.50/0 0 1994 American Chemical Society
pitfall of this type of determination arises from background chemiluminescence (CL) observed in the absence of fluorop h o r ~ . ~ -This I ~ problem can be circumvented by using the continuous addition of reagent (CAR) technique," thanks to its kinetic nature (rate measurements) and special way of mixing sample and reagents (continuous addition of a reagent solution at a constant rate to another solution containing the analyte to be determined). Thus, because the CAR technique effectivelysuppresses background emission, detection of species can be substantially improved relative to other static and flow systems used for handling samples and reagents. In this work, we chose the C L determination of phenothiazine derivatives in order to assess the implementation of peroxyoxalate chemiluminescence reaction by using the CAR technique; this is the first reported determination for these psychotropic drugs based on peroxyalate CL reactions. Thus, the bis(2,4,6-trichlorophenyl) oxalate (TCPO)/hydrogen peroxide system was used to develop a sensitive, rapid, and simple method for the determination of acepromazine, propiopromazine, thioridazine, promazine, trimeprazine, methotrimeprazine, and chlorpromazine. Phenothiazine derivatives are the most widely used drugs for treatment of psychiatric disorders;l2 these compounds are useful also in veterinary medicine for chemical restraint of animals for various diagnostic and clinical procedures as they exert a sedative effect by depressing the brain stem and connections to the cerebral cortex.13 On account of the sensitivity achieved in this work on the peroxyoxalate CL determination of acepromazine, as well as the widespread use of this phenothiazine in veterinary medicine (it reduces excitability so that animals can be easily handled), we chose this phenothiazine to apply the proposed CL method. Most recent methods for thedetermination of phenothiazine derivatives are based on liquid chromatography using various detection Although the lowest limits of detection (LODs) are generally provided by electrochemical detection, ~~
~~~
~~
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(7) Sigvardson, K.; Birks, J. W. Anal. Cfiem. 1983,55,432-435. (8) Weinberger, R.; Mannan, C. A.; Cerchio, M.; Grayeski, M. L. J . Cfiromotogr. 1984, 288,445-450. (9) Alvarez, F. J.; Parekh, N. J.; Matusqewski, B.; Givens, R. S.; Higveh, I.; Schowen, R. L. J . Am. Chem. SOC.1986, 108, 64356437. (10) Mann, B.; Grayeski, M. L. Anal. Cfiem. 1990, 62, 1532-1536. (1 1) Velasco, M.; Silva, M.; Ptez-Bendito, D. Anal. Cfiem. 1992,64,2359-2365. (1 2) Reynolds, J. E. F., Ed. Martindale. The Extra Pharmacopoeia, 29th ed.;The Pharmaceutical Press: London, 1989. (13) Booth, N. H. Psychotropic Agents. In Veterinary Pharmacology and Therapeutics, 6th ed.; Booth, N. H., McDonald, L. E., Eds.; Iowa State University Press: Ames, IA, 1988; Chapter 17. (14) Loennechen, T.; Andersen, A.; Hals, P. A,; Dahl, S. G. Tfier.Drug. Monir. 1990, 12, 574-581. (1 5 ) Loennechen, T.; Dahl, S. G. J . Cfiromatogr.1990, 503, 205-21 5 . (16) Keukens, H. J.; Aerts, M. M. L. J , Cfiromatogr.1989, 464, 149-161.
AnalflicalChemistry, Vol. 66,No. 22, November 15, 1994 4079
adsorption of the electroactive species can degrade the chromatographic efficiency for some drugs. Some gas chromatographicmethuds18provide LODs of a few nanograms per milliliter that can be lowered by at least 2 orders of magnitude using gas chromatography and mass spectrometry in tandem.lg Other recently reported alternatives to the determination of phenothiazine derivatives include (a) spectrophotometric methods,20,21which are widely used, even though they generally are less sensitive (pg/mL levels) and selective since most determinations are based on the phenothiazine oxidation and allow other reductants to be detected; (b) spectrofluorometric method^,^^-^^ many of which rely on the fluorescence of the corresponding oxidized forms (under relative mild conditions, phenothiazines are slowly oxidized by air to the corresponding sulfoxide^);^^ and (c) electrochemical method^,^^$^^ used for the determination of phenothiazines in biological fluids or pharmaceutical preparations, dependingon the sensitivity of the electroanalytical technique used.
EXPERIMENTAL SECTION Chemicals. All chemicalswere of analytical-reagent grade. All phenothiazines were obtained from Sigma Chemical Co. and used without further purification. Standard solutions containing 1000 mg/L of each were prepared by dissolving the required amount in acetone. A 1 t 2 M bis(2,4,6trichlorophenyl) oxalate solution was made by dissolving 449 mg of the chemical (Aldrich) in 100 mL of ethyl acetate. A 5X M tris(hydroxymethy1)methylamine (Tris; Merk) buffer solution was prepared by dissolving 151.4 mg of reagent in water and adding enough hydrochloric acid to adjust the pH to 9.0 in a final volume of 25 mL. It should be emphasized that some of the chemicals used are highly irritating and toxic, so they must be handled cautiously. Prolonged use of TCPO at a high concentration is not exceedingly troublesome, however, since replenishment and addition from the buret are both carried out automatically. Apparatus. The instrumental setup used is depicted in Figure 1. A Perkin-Elmer 650-10s spectrofluorometer with its light source switched off was used for chemiluminescence detection. A Metrohm Dossimat 665 autoburet, a magnetic stirrer, and a cylindric glass reaction vessel was also used. In order to allow the detector to acquire as much emitted light as possible, an Oriel 441 321 1-in.-diameter mirror was placed in front of the photomultiplier tube. Data were acquired and processed by a Netset PC-AT 16-MHz compatible computer ~
(17)McKay,G.;Cooper,J.K.;Midha,K.K.;Hall,K.;Hawes,E.M.J. Chromatogr. Biomed. Appl. 1982,22,417-422. (18)Rifai, N.;Howlett, C. M.; Levtzow, C. B.; Phillips, J. C.; Parker, N. C.; Cross, R. E. Ther. Drug Monit. 1988,IO, 194-196. (19) Jemal, M.; Ivashkiv, E.; Both, D.; Koski, R.; Cohen, A. I. Biomed. Environ. Mass Spectrom. 1987, 14,699-704. (20)Santoro, M. I. R. M.; Storpirtis, S.;Hackmann, E. R. M.; Magalhaes, J. F. Anal. Lett. 1989,22, 929-949. (21)Puzanowska-Tarasiewicz,H.; Karpinska,J.; Golebiewski, Z . Pharmazie 1989, 44,350. (22)Mellinger, T.J.; Keeler, C. E. Anal. Chem. 1964,36, 1840-1847. (23) Davidson, A. G. J. Pharm. Pharmacol. 1978,30,410-414. (24)Chen, D.; Rios, A.; Luque, de Castro, M. D.; ValcBrcel,M. Analyst 1991,116, 171-176. (25)Martfnez-Calatayud,J.; G6mez-Benit0,C. Anal. Chim. Acta 1992,256,105111. (26)Peng, T.;Yang, Z.; Lu, R. Yaoxue Xuebao 1990,25,277-283. (27)Tkach, V. I.; Clukhov, 0.I.; Tsyganok, L. P. Zh. Anal. Khim. 1991, 46, 1330-1 334.
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f
A
lml -9!ESb Signal
SPECTROFLUORIMETER
Flgure1. Schematicarrangementof the continuousadditionof reagent technique in peroxyoxalatechemiluminescence-based determinations.
equipped with a PC-Multilab 8 12 PG analog-to-digital converter. Data acquisition was synchronized with the start of the peroxyoxalate CL reaction by using a trigger acting simultaneously on the autoburet and the computer. All computations were performed by this processor using software written in MS-DOS QBasic version 1.1 (Microsoft, 19871993). CL Determination of Phenothiazines. A volume of up to 9 15 pL of standard phenothiazine solutioncontaining between -20 pmol and 200 nmol of the drug, 65 pL of concentrated hydrogen peroxide [33% (v/v) aqueous solution], and 20 pL of 5 X 1 t 2M Tris buffer of pH 9.0 was placed in the reaction vessel and accurately diluted to 1.0 mL with acetone. The reaction was developed by continuous addition of 1 X 1t2 M TCPO at a rate of 6.5 mL/min under continuous stirring. The CL signal was monitored throughout the reaction and kinetic data (relative CL intensity vs time) were acquired at a rate of 20 ms/point, the maximum reaction rate being measured in -1 s.
RESULTS AND DISCUSSION The nature of the background emission observed in peroxyoxalate CL reactions has been investigated by several authors. Because fluorescent impurities in the reaction medium are reported as an unlikely cause for the phen~menon,~ the formation and decomposition of CL reaction intermediates seems to be responsible for peroxyoxalate background CL. The background CL produced by various peroxyoxalate reagents with hydrogen peroxide was recently investigated by Mann and Grayeski.lo These authors have observed a broad emission band centered at -450 nm together with a weak emission band at 540 nm for the TCPO/hydrogen peroxide system. The spectra are time-dependent; thus, those species that emit light of a higher intensity and at -450 nm are formed relatively slowly. In addition, in excess hydrogen peroxide, only the intermediate peaks at -450 nm seem to prevail which exhibit little emission at the other wavelength. Accordingly, the detection of fluorophor-induced CL with minimum background emission entails use of excess of hydrogen peroxide (the second intermediate with a longer wavelength emission is not formed) and rapid recording of the CL intensity immediately after TCPO, hydrogen peroxide, and fluorophor are mixed owing to the relatively slow formation of the intermediate giving the peak at 450 nm.
12,
PHENOTHIAZINE
fl,
flz
fl3
ACEPROMAZINE
CO-CH,
H
C H r N ICH, )e
PROPIOPROMAZINE
CO-C8,
H
CHz-N ICH, i,
PROMAZINE
H
H
CHdJ
h
TRlMEPRAZlNE
H
CH,
CHrN
IC& h
METHOTRIMEPRAZINE
0-CH,
CH,
CH2.N f W i s
CHLORPROMAZINE
CI
H
CHrN I C 4 h
THIORIDAZINE
S-CH,
H
Q CH,
Figure 2. Structures of phenothiazine derivatives. nn "'"
I
0
I
0.5
1 .o
1.5
Time, seconds Figure 3. Relative CL intensityvs time plots for different phenothiazine derivatives: (1) propiopromazine, (2) acepromazine, (3) promazine, methotrimeprazine and trimeprazine, (4) thloridazine, and (5) chiorpromazine. (l),(2), and (4) 100 ng/mL; other phenothiazinederivatives, 250 ng/ml. (For experimentalconditions,see ExperimentalSection.)
-
In this work, these requisites can readily be met by using the CAR technique for development of the CL reaction. The CAR technique involves addition at a constant rate of one of the reagents involved from the autoburet. In principle, TCPO or hydrogen peroxide can be added; however, taking into account the above-described results, adding the TCPO from the autoburet resulted in higher signal-to-noise ratios because the hydrogen peroxide present in the reaction vessel was always in excess, which was mandatory in order to decrease background emission. In addition, the CAR technique used the maximum reaction rate (slope of the straight portion of the CL vs time curve at the start of the reaction) as the measurement parameter, which minimizes the effect of background emission since the intermediate involved is formed relatively slowly. It is worth emphasizing that the maximum reaction rate can be determined in only 1 s. In order to test the performance of the proposed approach, we chose seven phenothiazine derivatives, namely, acepromazine, propiopromazine, thioridazine, promazine, trimeprazine, methotrimeprazine, and chlorpromazine, the structural formulas of which are shown in Figure 2. Their CL vs time curves are shown in Figure 3, and as can be seen, the maximum response was exhibited by propiopromazine and acepromazine.
-
I12
0
1
6
7
o
8
PH
9
1
I
c
10
0
I
0.2
0.4
IH A
11
l
0.6
0.8
M
Figure 4. Effect of (A) pH and (B) the hydrogen peroxideconcentration on the maximum reactionrate: [acepromazine] = 100 ng/mL; [TCPO] = 0.01 M; addition rate, 6.5 mL/min.
Effect of Experimental Variables. On the basis of the above results, we chose acepromazine as the phenothiazine to be used in order to study the effect of experimental variables on the peroxyoxalate CL reaction. Both chemical and instrumental variables potentially influencing the system performance were investigated by using the univariate method. All concentrations quoted are referred to the final volume in the reaction vessel (1 .O mL) or in the autoburet. The effect of pH on the maximum reaction rate is shown in Figure 4A; as can be seen, a weakly basic solution (pH 8.0) ensured the maximum analytical signal, so it was chosen as optimal and adjusted with a Tris buffer solution. The concentration and pH of this buffer solution were also investigated, the water content in the reaction medium being critical since the CL signal increased with decreasing water content. Thus, in a reaction medium consisting of 91.59.5 acetonelwater, where the water would be exclusively supplied by the hydrogen peroxide and buffer added to the reaction vessel, use of Tris buffer of pH 9.0 ensures the present working pH (8.0) in the reaction vessel thanks to the high acetone content in the reaction medium. The effect of the Tris buffer concentration was evaluated in the range 1.0 X 104-4.0 X M; the maximum reaction rate peaked at 1.0 X M, above and below which it decreased. This concentration was obtained by placing 20 pL of 5.0 X M Tris buffer of pH 9.0 in the reaction vessel. The effect of the hydrogen peroxide concentration was studied over the range 0.07-0.7 M. As can be seen from Figure 4B,the maximum reaction rate was reached at -0.5 M, above which it remained virtually constant. A 0.7 M concentration was thus chosen (65 KL of concentrated hydrogen peroxide was placed in the reaction vessel), which ensured a large excess of reagent relative to TCPO in order to suppress background emission. It is noteworthy that the maximum TCPO concentration that could be used without solubility problems was 0.01 M, which was added from the autoburet and therefore diluted in the reaction vessel. One of the most important variables affecting the peroxyoxalate CL reaction was the nature and concentration of mixed aqueous/organic solvent used as the reaction medium. Taking into account that TCPO is most stable and efficient in organic solvents such as ethyl acetate, which are usually immiscible with water, a cosolvent was required to improve its water miscibility. This problem is also encountered when the peroxyoxalate CL reaction is used for detection in liquid Analytical Chemistty, VoL 66,No. 22, November 15, 1994
4081
Table 1. Influence of Cosolvents on the Peroxyoxalate CL Reactlon mixed aqueous/organic solvent Composition, % max reaction water acetone acetonitrile ethanol rate, X l W 3 s-’
8.5 12.5 17.5 22.5 27.5 32.5 31.5 8.5 12.5 8.5 12.5 8.5 8.5 8.5 8.5 8.5 8.5
91.5 87.5 82.5 77.5 72.5 61.5 62.5 91.5 87.5 91.5 87.5 35.5 56.0 56.0 35.5
56.0 35.5 56.0 35.5
35.5 56.0 35.5 56.0
10.3 6.5 4.9 4.1 4.0 4.5 5.3 8.4 5.3 7.5 4.5 9.o 9.6 8.4 6.3 5.8 5.8
c
a
12
-
10
I
mI 0 X
C
a 6
.-0
U
0
2
4 2
5 E
0
;5
-2
.-
E
Figure 5. Responsesurface for the influenceof the TCPO concentration and Its rate of addiiion from the autoburet: [acepromazine] = 100 nglmL; [H202]= 0.7 M; pH, 8.0.
chromatography.2g In other cases (e.g., the stopped-flow experiments of Givens et al.29) the optimum water content can be as high as a 40%, due to a compromise between the maximum CL intensity achieved and the reaction rate since the time required for the maximum increases and the CL decreases with increasing the water content. In order to study the effect of this variable by using the CAR technique, the water content was kept at a minimum (8.5%) because of the volumes of Tris (20 pL) and hydrogen peroxide (65 pL) added to the reaction vessel (1 .O mL). Acetone, acetonitrile (two organic solvents typically used in this peroxyoxalate CL reaction), ethanol, and mixtures of the three were tested as cosolvents; the results obtained are shown in Table 1. Note that percentages are referred to the initial aqueous/organic composition in the reaction vessel prior to addition of the TCPO/ethyl acetate solution. Ascan be seen, the best results was provided by a 91.553.5 (v/v) acetone/water medium, where, even through the straight linear portion of the CL vs time curve lies in the millisecond range, the CAR technique provides highly reliable and accurate measurements of the maximum reaction rate. One important consideration in adapting peroxyoxalate CL detection to CAR technique is the concentration of the TCPO solution and its rate of addition from the autoburet, the effects of which were investigated over the ranges 1.0 X lO”-l .OX M and0.5-8.0 mL/min, respectively. Because these two variables are closely related (the actual TCPO concentration in the reaction vessel depends on both), their influence on the maximum reaction rate is illustrated by the response surface shown in Figure 5 . As can be seen, the effect of both variables on the CL reaction was similar, even though the addition rate reached a near-zero-order kinetic region above 6.5 mL/min. This addition rate and the maximum concentration of TCPO tested (0.01 M in ethyl acetate) were thus selected for subsequent experiments. Determination of Phenothiazine Derivatives. Working curves of maximum reaction rate vs concentration were run for each of the phenothiazine derivatives shown in Figure 2. (28) Weber, A. J.; Grayeski, M. L. Anal. Chem. 1987, 59, 1452-1457. (29) Givens, R. S.;Jencen, D. A,; Riley, C. M.; Stobaugh, J. F.; Chokshi, H.; Hanaoka, N. J . Pharm. Biomed. Anal. 1990,8, 477-491.
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Table 2 gives the least-squares parameters for the working curves and the detection limits, calculated according to the IUPAC’s recommendati~ns.~O As can be seen, the maximum sensitivity was exhibited by propiopromazine and acepromazine. The precision, expressed as the relative standard deviation (RSD), was determined by analyzing 11 samples containing a concentration within the range of the working curve for each phenothiazine. The RSD for the maximum reaction rate was 1.0-3.7596. Another analytical figure of merit for this peroxyoxalate CL determination of phenothiazine derivatives is the sample throughput, which was calculated from the time needed to perform three replicate analyses including sample changeover in the CAR system. The throughput thus obtained was -90 samples/h, which is more than adequate for routine analyses of phenothiazines. The proposed method for acepromazine and propiopromazine, the two phenothiazines exhibiting the highest sensitivity, is a useful choice for the determination of these drugs as it provides a broader dynamic range and a higher sensitivity than existing spectrofluorometric3* and enzyme-linked immunosorbent assay (ELISA)32 methods (typical LODs of a few micrograms per milliliter), as well as high-performance liquid chromatographic (HPLC) m e t h o d ~ l ~ 3using ~ ~ J spec~ trofluorometric or amperometric detection (LODs of 3-60 ng/mL). Also, the precision of the proposed method is similar to or even higher than that of such methods, as is its throughput, because of the expeditiousness with which the CAR technique allows one to obtain analytical information. Interferences. As stated in the introduction, peroxyoxalate CL determinations are quite selective because not all fluorophors are equally efficient as energy acceptors. In order to verify this assertion, a study of potential interferents (different drug types) was carried out. Whether such compounds would (30) Long, G. L.;Wincfordner, J. D. A w l . Chcm. 1983.55, 712A-724A. (31) Navas-Diaz, A. Anal. Chim. Aero 1991, 225, 297-303. (32) Smith, M.L.; Chapman, C. B. Res. Vet. Sei. 1987, 42. 415-417. (33) Haagsma, N.; Bathelt, E. R.; Engelsma, J. W.J . Chromarogr. 1988, 436, 73-79. (34) Etter,R.;Battaglia,R.;Noser,J.;Schuppisser, F. Mitt. Geb.Lebemmitrefunters. Hyg.1984, 75,441-458.
Table 2. Flgures of Merlt of the Callbratlon Graphs and LODs Obtalned In the CL Determlnatlon of Phenothlazlnes
calibration plot phenothiazine acepromazine propiopromazine thioridazine promazine trimeprazine methotrimeprazine chlorpromazine
mol/L 2.3 X 2.6 X 9.8 X 6.2 X 6.7 X 2.9 X 7.0 X
10-8-9.0 10-8-1.1 10-8-7.4 10-7-1.2 lt7-2.1 1p7-1.8 1k7-8.4
X X X X X X X
lk5 10-4
10-4 10-4 10-4 le5
ng/mL
linear regression equation"
(7.5-29.5) X lo3 (9.0-36.0) X lo3 (36-27.3) X lo3 (180-35.5) X lo3 (200-64.0) X lo3 (96-59.1) X 103 (220-27.0) X lo3
mrr = 5.8 X 10-4 4.5 X 104C mrr = -1.0 X 1 0 4 + 5.4 X 104C mrr = -9.0 X + 4.5 X 103C mrr = -6.0 X + 2.2 X 103C mrr = 1.5 X 10-4 + 2.5 X 103C mrr = 5.3 X 10-4 + 1.7 X 103C mrr = 6.7 X 10-4 + 1.8 X 103C
+
corr coeffb
LOD, ng/mL
0.9973 0.9984 0.9986 0.9987 0.9971 0.997 1 0.9968
2.2 2.7 10.9 54.5 60.6 29.1 66.5
mrr, maximum reaction rate; C, molar concentration. n = 16.
~~
Table 3. Influence of Forelgn Drugs on the CL Determlnatlon of 75 ng/mL Acepromazlne
drug noradrenaline, oxazepam, bromazepam, triazolam, clotiazepam, lormetazepam, cephalexin, atropine, dopamine, ketamine, morphine, methadone, pentobarbital, xylazine, desipramine, imipramine, trimipramine, amitriptyline, nortriptyline, 5methyltryptamine, N-methyltryptamine, harmine nitrazepam adrenaline promethazine, chlorpromazine, perphenazine promazine, methotrimeprazine propiopromazine, trimeprazine, thioridazine
tolerated ratio 100
50 10 7.5 2.0 acepromazine > thioridazine > trimeprazine > promazine > chlorpromazine > methotrimeprazine which is mainly a function of the R1 radical in the structure of each phenothiazine. In order to account for such as dependence, the effect of substituent R1 on the reactivity of the phenothiazine derivatives tested in the CL reaction was studied for acepromazine, propiopromazine, promazine, and chlorpromazine, which differ in the structure of their R1 radical only. The comparison was based on the resonance and field parameters usually employed in correlation analyses of
chemical data.35.36The reactivity, expressed as the logarithm of the slope of the calibration graph, was found to be closely related to factor ur, which accounts for resonance, but not to ui, which accounts for inductive effects. Therefore, the observed reactivity differences between the phenothiazine derivatives seemingly arises largely from the contribution of substituent R1 to the molecular resonance. To examine the nature of the fluorophor involved in this CL reaction, various spectrofluorometric experiments were carried out. The fluorescence spectrum for each phenothiazine was recorded at a pH of 8.0 under the selected experimental conditions, both in the presence and in the absence of hydrogen peroxide. Both series of spectra exhibited the same intensity and excitation and emission wavelengths, which suggests that phenothiazine is probably not oxidized to its sulfoxide. This is consistent with previously reported results: the oxidation of phenothiazine derivatives by hydrogen peroxide requires 50% acetic acid and heating in a boiling water bath for 10 ~ n i n . ~ 'Therefore, phenothiazine itself seemingly acts as fluorophor in the TCPO/hydrogen peroxide CL system. Determination of Acepromazine in Horse Plasma. The above results suggested the feasibility of applying the proposed method to the analysis of acepromazine in real biological samples. For determination of the drug in horse plasma, 1.O mL of sample was mixed with 1.0 mL of 0.2 M sodium carbonate buffer (pH 10.6) and 10 mL of 99:l (v/v) hexane/ butanol in a separation funnel. The mixture was shaken for 15 min, and the separated organic phase was then evaporated to near-dryness. The residue was dissolved in acetone and collected in the reaction vessel, after which it was subjected to the above-described procedure. Prior to this treatment, the horse plasma was spiked with different known amounts of acepromazine since the compound is largely detected unaltered in plasma.38 As can be seen in Table 4, the spiked amounts ranged from 15 to 300 ng (1 mL of horse plasma sample) since the initial plasma levels of the drug after a typical dosing with -0.3 mg/kg are in the region, of 100 ng/mL but fall rapidly to -25 ng/mL within 30 min. Later, the plasma levels of the drug dropped more slowly (the concentration was -10 ng/mL after -8 h.).38 The LOD obtained by using the proposed method is low enough for the acepromazine content in these clinical samples to be measured, as well as for development of pharmacokinetic studies.
-
(35) Hansch, C.; Leo, A.; Taft, R . W . Chem. Rev. 1991, 91, 165-195. (36) Exner, 0.Correlarion Analysis of Chemical Data; Plenum Press: New York, 1988; Chapter 5 . (37) Ragland, J. B.; Kinross-Wright, V. J. Anal. Chem. 1964, 36, 1356-1359. (38) Tobin, T. Drugs and rhe Performance Horse; Charles C. Thomas Publisher: Springfield, IL 1981; pp 240-242.
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Table 4. Recovery of Acepromazlne Added to 1.0 mL of Horse Plasma sample added, ng found, ng recovery, % 1 2 3 4
5
15 50 100 200 300
15.3 48.5 101.7 188.8 286.8
102.0 97.0 101.7 94.4 95.6
The recoveries obtained, from 94.4 to 102.096, are given in Table 4 and testify to the good performance of the proposed CL method in the determination of acepromazine in this type of sample. To further evaluate the consistency of the results, the calibration graphs run for the proposed reaction medium and the horse plasma samples were fitted by least-squares regression. The regression equation and statistics obtained were as follows: Cp = (-2.16 f 1.33) X lo4 S, = 1.35 X lo-'
+ (1.09 f 0.02)Cs r = 0.9996
where C,, and C, denote the concentration (mol/L) of acepromazine found obtained by using the calibration graph in plasma and the proposed reaction medium, respectively, and S, denotes the standard error of the estimate. The statistical parameter values found confirm the high degree of correlation between the two methods and hence the absence of matrix effects on the determination.
CONCLUSIONS In this work, the continuous addition of reagent technique was shown to be a useful means for the removal of background
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
emission in peroxyoxalate chemiluminescence determinations. This ability arose from the special way in which the sample and reagent are mixed in the CAR technique, in addition to its kinetic connotations. The proposed approach, based on the TCPO/hydrogen peroxide system, was evaluated in the CL determination of phenothiazines, which is reported for the first time. The study of the influence of variables revealed virtually the same dependence observed in other batch and flow systems, except for two distinct features: (1) higher hydrogen peroxide/TCPO concentration ratios can be used as a result of the TCPO solution being added to the reaction vessel containing the oxidant and kinetic data being acquired simultaneously (these conditions favor suppression of background emission), and (2) there is nodead time between mixing TCPO and hydrogen peroxide and reaction with the fluorophor (incontrast with those flow systems whereTCPO and hydrogen peroxide are generally mixed in a first instance and the mixed flow is reacted with the fluorophor then), which increases the efficiency of the peroxyoxalate CL reaction and avoids the typical instability of aqueous TCPO solutions. On the other hand, the detection limits and working ranges found are quite acceptable for analysis of the drugs in real samples.
ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Direcci6n General Interministerial de Ciencia y Tecnologfa (DIGICyT) for the realization of this work as part of Project PB9 1-0840. Received for review March 23, 1994. Accepted June 27, 1994."
* Abstract published in Aduance ACS Abstracts, September 15, 1994.
