time is normally around 30 s, so that one usually waits a few minutes before perturbing the system again to ensure that all has reequilibrated. Also, a solution is used, generally, only once or twice before changing. Fresh degassed solution is kept in the circulating constant temperature bath ready for use. This apparatus using typically from 8 to 10 kV perturbation voltage is capable of studying reactions whose relaxation times are longer than 300 ns. This and a 1-cm optical path greatly extend the range of reaction rate constants that may be obtained from the capacitor discharge method.
ACKNOWLEDGMENT The authors thank Paolo Priarone for the cell construction and for assistance in the system integration.
LITERATURE CITED (1) G. Gzerlinski, Rev. Sci. Insfrum., 3 3 , 1184 (1962). (2) A. D. Yu, M. D. Waissbluth, and R. A. Geieger, Rev. Sci. Insfrum., 44. 1390 (1973). (3) E. Caldin, Cbem. Br., 11, 4 (1975). (4) T. C. French and G. G. Hammes, Methods Enzymoi.. 16, 5-21 (1969). (5) G. W. Hoffman, Rev. Sci. Instrum., 42, 1643 (1971). (6) S. Ameen, Rev. Sci. Instrum., 46, 1209 (1975). (7) G. Czerlinski and A. Weiss, Appi. Opf., 4, 59 (1965). (8) “Photomuttiplier Manual”, (RCA Corp., New Jersey, 1970) Technical Series PT-61, p 62. (9) S. Fich, “Transient Analysis in Electrical Engineering”, Prentice-Hall, Englewood Cliffs, N.J., 1951, pp 77-78. (10) J. R. Sutter, M. Krishnamurthy, and P. Hambright J. Chem. Soc, @em. Commun., 13 (1975).
RECEIVED for review January 19, 1977. Accepted March 24, 1977.
Simplified Gas Chromatographic Method for the Determination of Chlorpheniramine in Serum James W. Barnhart’ and James D. Johnson’ Health and Consumer Products Department, The Dow Chemical Company, Indianapolis, Indiana 46268
An electron capture gas chromatographic method for the determination of brompheniramine in blood has been reported ( I ) . Permanganate oxidation was used to produce p bromophenyl-2-pyridyl ketone, the chromatographed species. As the authors noted, it would have equal utility for the determination of chlorpheniramine. Partition chromatography was used to remove two interfering metabolites, the monoand didemethylated compounds. Acetylation was thought to be inapplicable as a means of separating these metabolites. We have found that acetylation can be used for the removal of comparable metabolites of chlorpheniramine, resulting in a considerable saving of time.
A
lw
H C CH2CH2N(CH3)2
@
A
K Mn04
0 H-
v
-6
EXPERIMENTAL Instrumentation. A Packard Model 7300 gas chromatograph equipped with a tritium electron capture detector was used for the analyses. A 6 ft X 3 mm silanized glass column was packed with 3% SP-2250 on 100-120 mesh Supelcoport. Detector and injector temperatures were 225 “C and the oven temperature was 195 “C. Nitrogen flow was 70 mL/min and the detector voltage setting was 20 V. Procedure. An appropriate quantity of brompheniramine (internal standard) was added to 2.0 mL of plasma or serum in a test tube and mixed with 0.2 mL of 10 N KOH. The samples were heated in a boiling water bath for 10 min. After cooling, the samples were mixed vigorously for 1-2 min on a rotary mixer with 4 mL of diethyl ether (Fisher anhydrous). After centrifugation, the ether layer was transferred to a second tube and mixed with 1 drop of acetic anhydride (Fisher reagent grade, redistilled). After 5 min, 1.0 mL of 0.1 M pH 5.5 citrate-phosphate buffer was added and mixed vigorously for 1-2 min. After centrifugation,the ether layer was discarded. The aqueous layer was then washed twice with 3-mL portions of ether. All traces of residual ether were removed by placing the samples under a stream of nitrogen for 15 min in a 100 “C sand bath. After adding 0.15 mL of 2 N KOH and 1.5 mL of 1%KMn04,the tubes were stoppered loosely with marbles and heated in a boiling water bath Present address, 3M Company, St. Paul, Minn.
Table I. Human Serum Chlorpheniramine Levels After Single Oral Dose Time, Serum chlorpheniramine, ng/mL as the free base h 4-mg dose 8-mg dose 12-mg dose 0
0.5 1.0 1.5 2.0 4.0 8.0 12 24
0.1 0.6 2.1 3.7 4.5 5.5 4.3 3.7 2.2
f
0.0 (SD) 0.2 1.0 1.2 1.1
t
1.0
t
0.5 0.6
i i
t
*
* 0.7 i
0.3 i 0.6 i 3.2 t 6.0 t 7.2 i 10.4 i 8 . 3 ?: 6.7 i 4.1 ?:
0 . 4 (SD) 0.8 3.1 4.4 3.7 2.6 2.4 1.6 1.3
0.1 i 1.4 i 6.7 t 13.2 t 15.3 i 17.6 i 14.7 i 12.8 t 8 . 6 ?:
0 . 1 (SD) 1.2 4.6 6.7 4.5 5.1 3.9 4.8 4.7
for 10 min. The cooled samples were extracted with 1 mL of
isooctane (Mallinckrodt Nanograde) on a rotary mixer. After evaporation of the solvent, the residue was dissolved in 25-50 pL of isooctane containing 5% acetone. Standards were taken through the entire procedure and peak height ratios plotted vs. ng of chlorpheniramine. All reagents were reagent grade and double glass-distilled water was used throughout.
RESULTS AND DISCUSSION Since the permanganate oxidation results in the same product whether chlorpheniramine or the demethylated metabolites are the starting material, it is essential that the latter be efficiently removed. Even though a pyridyl moiety remains in the resultant acetamide, it is a very weak base and does distribute to the organic phase a t the pH employed. Ionization constants for chlorpheniramine are 9.2 and 4.0 for pKa, and pKaz,respectively (2). The ionization constants of the acetylated metabolites are unknown. Assuming they are of the same magnitude as pK, of chlorpheniramine, significant protonation would not be present at pH values >5. This was confirmed by determining the degree of interference of both metabolites under different pH conditions (Figure 1). The stated pH was the pH of the original buffer. The actual pH during the extraction was slightly lower because of the acetic anhydride added earlier. Interference was less than 1% a t pH values of 4 or above. In another experiment, quantities of both demethylated metabolites ranging from 0.25-5 pug were added to control plasma and carried through the analysis. Less than 1% of these amounts was recovered as apparent ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
1085
I51
3.0
3.5
4.0
4.5
5.0
5.5
PH
Interference of two chlorpheniramine metabolites, the mono(V)and didemethylated (0)compounds, under different pH conditions Figure 1.
chlorpheniramine, about 0.1% of the secondary amine and about 0.6% of the primary amine. Under actual conditions, the metabolites are present in quantities of about the same order of magnitude as chlorpheniramine, so this type of efficiency is more than adequate. Human control plasma containing 10.0 ng/mL (calculated as the free base) of chlorpheniramine maleate was analyzed using the procedure given above. The mean f SD obtained from ten replicates was 10.0 f 0.52. The sensitivity of the procedure is adequate for the determination of as little as 0.5-1.0 ng/mL of drug.
The need for clean glassware and pure reagents cannot be overemphasized. Other potential problem areas are the evaporation of ether just before the oxidation step and loss of sample in nonpolar solvents. If the permanganate is depleted during the oxidation, it is almost certain that some residual ether was present and more stringent measures must be instituted for its removal. The other potential problem is adsorption of the ketone during storage of the sample in nonpolar solvents. This can be prevented by adding a small amount of acetone to the sample. Some typical results are given in Table I. Human volunteers received a single dose of chlorpheniramine maleate and blood samples were taken at intervals through 24 h. There were four subjects in each group. Based on these results, the terminal half-life in serum was 18 h. ACKNOWLEDGMENT The authors thank Ms. P. A. Huwel for her valuable technical assistance. The mono- and didemethylated metabolites were a generous gift of the Smith Kline and French Laboratories, Philadelphia, Pa. LITERATURE C I T E D (I) R. B. Bruce, J. E. Pins, and F. M. Pinchbeck, Anal. Chem., 40, 1246
(1988). (2) T. D. Doyle and J. Levine, J . Assoc. Off. Anal. Chem., 51, 191 (1968)
RECEIVED for review February 25,1977. Accepted March 23, 1977.
Precolumn Synthesis of Trimethylsilyl Derivatives of Aqueous Phosphate For Gas Chromatographic Analysis Rlchard H. Getty, Julia Stone,' and Richard H. Hanson" Chemistry Department, University of Arkansas at Little Rock, Little Rock, Arkansas 72204
The synthesis of volatile derivatives from nonvolatile samples frequently is performed prior to analysis by gas chromatography. When this process can be accomplished on the column or in a precolumn, the speed of analysis is faster and smaller samples can be analyzed. Several authors have studied the on-column silylation process. Esposito (1) showed that by using an on-column synthesis technique, it was possible to silylate compounds dissolved in reactive solvents such as water or alcohol. The concentration of the samples was between 1 and 10%. No data were presented to indicate if the reactions were quantitative. Morrow (2) constructed a precolumn assembly where solvent vapor was purged to the atmosphere prior to silylation. All volatile reaction products were swept onto the column and analyzed. Conversion to the orthophosphate derivative was less than 50% with a 48 pg sample. Matthews (3) was able to lower the detection limit, on orthophosphate to 1 pg, but was unable to reproduce peak areas. The peaks were poorly shaped with serious tailing. Wiese and Hanson (4) quantitatively silylated orthophosphate in a precolumn in the 10-100 pg range. Above the upper limit, conversion was not complete. This report describes a technique which was used to quantitatively analyze aqueous orthophosphate in the 0.25-5 pg range using a precolumn reaction system. The problems of peak shape, tailing, and reproducibility were minimized. 'Present address, Chemistry Department, University of Indiana, Bloomington, Ind. 47401 1086
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
Some organic acids were also successfully silylated and analyzed. The technique of reaction gas chromatography was applied to the problem of silylating aqueous inorganic anions to simplify the reported procedures (5,6). In both reports, the anion was converted to the ammonium form by an ion exchanger prior to synthesis. The first report required 10 mg of salt dissolved in a nonaqueous solvent. Silylation was accomplished overnight in a reaction vial and 125 pg of the salt injected for analysis. In the second paper, silylation was accomplished in a much shorter time. Two hundred mL of 0.1 part per million orthophosphate were concentrated, silylated, and analyzed, As little as 0.02 pg of phosphate were detected. In the technique developed and described below, much less volume of sample was required. Quantitative conversion of 0.25 pg of orthophosphate in 5 pL of sample was possible without treatment with an ion exchanger or concentrating into a nonaqueous solvent. EXPERIMENTAL Apparatus. A Hewlett-Packard 5750 gas chromatograph was
equipped with dual flame ionization detectors. The analytical column was 6-f00t, '/.,-inch glass packed with 5% OV-225on 60/80 mesh Chromosorb W, DMCS treated, and acid washed. The flame detector was operated at 250 "C, the injection port at 150 "C, and the column at 135 "C. Nitrogen carrier gas flow was 60 mL/min at 50 psi. The precolumn was the commercially available Pyrolysis Sampling System from Hamilton Company. This accessory, though designed as a pyrolysis attachment, was easily adapted
