[CY-] for Cd(II), computed by means of Equations 8 and 8', 9 and 9', are plotted LIS. -log CM, and are compared with the experimental values. In Figure 2 , the same plots are reported for bromide ion by using the corresponding value of K ( = lo8). In Figures 3 and 4, theoretical and experimental values are reported for Zn(I1) and Ni(I1). The values of the over-all formation constants used in = 5 . 5 ; log 8 2 = the calculation are ( 8 ) , for Cd(I1): log 10.6; log p 3 = 15.3; log p4 = 18.9. For Zn(I1): log p 2 = 11.0; log p3 = 16.0; log p4 = 19.6. For Ni(I1): log p4 = 30. The formation of hydroxo complexes was not considered, since they are negligible in the p H range studied (8). The same experiments performed with a chloride membrane electrode were not reproducible and the measured values are not stable and are very sensitive to stirring and to stray currents. The experimental values confirm generally the theoretical treatment; furthermore, if it is considered that diffusion coefficients differences through the diffusion barrier at the electrode surface were not taken in account and that large variations in the pi values are reported in the literature, the agreement between experimental and theoretical values is satisfactory. In the case of Cd(II), with the AgBr electrode, an inflection at 1:4 meta1:ligand molar ratio can be seen (Figure 2 ) : and experimental points do not fit the theoretical curves, probably for a slow kinetics a t the electrode surface of the reaction between AgBr and the complex species in the solution.
Also for Ni(II), a discrepancy can be observed with a metal excess. This can be due to the formation of intermediate complexes, the stability constants of which are not available. Furthermore, colloidal species could be formed in solution; in fact, titrations cannot be performed a t C C N = lO-*Mowing to a precipitate formation. From the curves obtained, it is concluded that halide membrane electrodes can be used in direct potentiometry as a sensor either of total cyanide or of free cyanide, depending on the strength of the complexes in solution and on the metal concentration. In the presence of weak complexes, the AgI and, better, the AgBr electrode becomes an indicator of total cyanide. I t can be theorized that the metal complexes react as well as free cyanide with AgI or AgBr to give I - or Br- determining the electrode potential. In the presence of strong complexes, AgI more than AgBr becomes an indicator of free cyanide in a large range of pCN, but when the metal ion is in excess, the electrode potential is no longer an indicator of free cyanide in solution. The failure of the chloride membrane electrodes to give stable potentials in cyanide solutions can be explained according to the kinetic experiments reported (9) by which the dissolution rate a t the phase boundary is so large that the concentration of the complexing agent is considerably depleted a t the surface. A large dissolution rate explains well the sensitivity to stirring and to pretreatment of the membrane.
(8) L . G. Sillen and A. E. Martell, "Stability Constants," Chem. SOC. Spec. Pub/., 17and 25,1964-1972. (9) W. Jaenicke, Z.Elektrochem., 55, 648 (1951).
Received for review May 31, 1973. Accepted September 24, 1973.
Quantitative Analysis of a Multicomponent Drug Product Using Liquid Chromatography J. S. Mayell, C. F. Hiskey, and Leon Lachman Endo Laboratories, Inc., E. 1. du Pont d e Nemours, 1000 Stewart Avenue, Garden City, N. Y. 11530
This investigation was undertaken to find a quick and accurate stability-indicating method for the determination of several active ingredients present in an analgesic combination drug product. Several of the analgesic ingredients are thermally unstable because of the presence of active hydrogen on the carbonyl and phenolic groups. In gas chromatographic analysis, because of very high temperature (200 to 300 "C), many compounds decompose. Liquid chromatography where the products generally do not degrade because of moderate temperature (usually ambient to about 40 "C), and are detected immediately after separation from a column, was thus the logical technique to choose. Ion exchange columns, using aqueous solvent, were tried as most of the active ingredients are ionic and water-soluble. It should be pointed out that not all retardations by ion exchange columns are purely ionic, they may also occur by other interactions between the resin substrate and the molecule under investigation. Several investigators (1, 2 ) have studied the separation of analgesic drug combinations, (1) R A H e n r y and J A. Schmidt, Chromafographm 3, 116 (1970) (2) R L. Stevenson and C A. Burtis. J Chromafogr, 61, 253 (1971).
using anion exchange columns. They were not satisfactory as caffeine eluted with the solvent front. Strong cation exchange resin (SCX) and Chelex 100 loaded with copper(I1) ion (elution by aqueous ammonia) ( 3 ) , could not resolve the aspirin and solvent peaks. I t was also not possible to separate the hexobarbital from the phenacetin peak when a SCX column was employed. Separation of the analgesic ingredients using weak anion exchange (WAX) resin was quite successful and its use for the quantitative determination of caffeine, aspirin, hexobarbital, and phenacetin will be presented.
EXPERIMENTAL Materials. The weak anion exchange (WAX) column contains "Zipax" chromatographic support coated with an amine substituted polyamide polymer (Du Pont product No. 820960010). Zipax ( 4 ) consists of spherical glass microbeads nominally 30 microns in diameter with a thin porous crust approximately l micron thick. These beads have a surface area of about l m*/gram, (3) J . C. Wolford. J . A. Dean, and Gerald Goldstein. J. Chromatogr., 62, 148 (1971). (4) J. J . Kirkland, Anal. Chem., 43 (12), 36A (1971).
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 3, M A R C H 1974
449
C
b
Figure 1. Effect of nitric acid on the separation of caffeine (C)
and aspirin
(A)
1
5
IO 15 TIME- MINUTES
20
25I
Figure 3. Effect of solvents (water and methanol), oxyc:odone
- ROOM
(OxyC) and homatropine ( H O M ) on the caffeine (C) peak
TEMP.
Table I. LC P a r a m e t e r s Instrument Column
I .t
n
Mobile phase
i3
Temperatures
s
Pressure Flow rate Injection volume Detector Chart speed Disc Integrator TIM,
(A)
are freeflowing and can be packed easily. Percodan tablets and Percobarb capsules are drug products of Endo Laboratories, Garden City, N.Y. The aspirin, phenacetin, caffeine, and hexobarbital standards used conform to USP specifications. The internal standards, benzoic acid for Percodan and methylparaben for Percobarb were of analytical grade from Eastman Kodak. Sodium sulfate anhydrous, reagent grade of J . T. Baker Chemical Co., Phillipsburg, N.J., was used. Equipment. Liquid chromatograph Model 820 LC of Du Pont was used throughout the study. The detector was a precision 254-nm fixed wave length photometer. The stainless steel column was 1 meter long with an internal diameter of 2.1 mm.
RESULTS AND DISCUSSION
To analyze multicomponent analgesic drug products, the existing techniques of spectrophotometry and gas chromatography take considerable time because of several separations necessary in these procedures. Liquid chromatography used in this study involves practically no prior separation procedure. Oxycodone, homatropine, caffeine, aspirin, hexobarbital, and phenacetin are the active ingredients in the two drugs under investigation. Hexobarbital is present only in Percobarb capsules. Several salts of citrates, perchlorates, and phosphates were tried in the mobile phase but anhydrous sodium sulfate was found best for effective resolution of the peaks. The higher the concentration of sodium sulfate, the better the separation of methanol and caffeine. After careful study, 1.5M Na2S04 was selected since a t higher concentrations it could clog the narrow bored tubes owing to crystallization, especially in winter. The presence of nitric acid in the mobile phase is very important to separate the caffeine and aspirin peaks as seen in Figure 1. The concentration of nitric acid used is 450
uv
5.0 min 'inch
On
MINUTES
Figure 2. Effect of temperature to separate hexobarbital (HB)
and aspirin
DuPont 820 LC 1.0 meter, 2.1-mm i.d., weak anion exchange (WAX) 1.5M Na2SOI;5.0 X 10-3M HNOj 40 OC-column 50 OC-reservoir 1000 psig 0.7 m1,'min (Direct measurement) 1.2 mlimin (Rotometer) 5 rl
significant since, a t higher concentrations, it causes the aspirin peak to move toward the phenacetin peak while a t lower concentrations, it becomes difficult to resolve the caffeine and aspirin peaks. An optimum concentration of 5.0 X 10-3M nitric acid was established for best resolution. When 4 x 10-2M nitric acid alone was used as the mobile phase, the aspirin peak eluted after the phenacetin peak. A slight increase in temperature from ambient to 40 "C, played an important role in separating one of the constituents. Figure 2 shows chromatograms of a Percobarb sample at room temperature and at 40 "C.At room temperature, the hexobarbital peak is buried in the tailings of the aspirin peak. When the temperature was increased to 40 "C,a well developed peak of hexobarbital (in between the aspirin and phenacetin peaks) was observed. This pronounced beneficial effect a t the higher temperature may be due to change in the partition coefficient of aspirin and/or hexobarbital in the mobile phase. The hump in the caffeine peak a t room temperature is due to the solvent methanol. This, however, does not occur a t 40 "C. By working a t high pressures due to increases in flow rate, analysis time is shortened. However, peak resolution, especially in these multicomponent drug products, decreases as the pressure is raised. A compromise at 1000 psig with an analysis time of about 25 minutes was arrived a t for this investigation. Under optimized conditions, it is possible to determine free salicyclic acid (FSA), the hydrolyzed product of aspirin, as it elutes after the phenacetin peak. However, due to the low absorptivity of FSA a t 254 nm and the small concentrations (less than 1%) that usually exist. the present system is not suitable for the quantitative determination of FSA. The use of wavelengths other than 254 nm and complex compound formations are other possibilities
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 3, M A R C H 1974
Table 11. Quantitative Data-Area
us.
Amount determined, mg. Actual amount
Peak ht
A mg
P 192.0 mg
C 32.0 mg
Accuracy,
Area
189.1 178.0 182.8 185.0 194.2 187.5 38.1 29.9 35.6
180.0
Peak Height 70
Peak ht
Area
5.1 1.1 1.5 3.7 1.1 1.3 19.1 6.4 11.3
1.4 0.1 0.1 2.1 3.3 1.7 0.9 0.9 1.6
182.6 180.2 178.8 187.9 185.5 186.6 31.7 31.7 31.5
for FSA determination. An advantage of this technique for the simultaneous determination of aspirin and FSA is that it could be used effectively to study the kinetics of decomposition of aspirin by water. In order to investigate the effect of solvents (water and methanol), oxycodone (OxyC) and homatropine (HOM) on the analysis of other constituents, the chromatograms were obtained at very low pressure of 230 psig (0.15 ml/ min), as seen in Figure 3. Water and methanol (curves B and C) do not interfere with the caffeine peak (curve A ) . OxyC and HOM elute with the solvent front. Three times the normal dosage of OxyC (0.27 mg/ml us. normal dosage of 0.09 mg/ml) and HOM (0.024 mg/ml us. normal dosage of 0.008 mg/ml) under our experimental conditions showed no interference with the caffeine peak, curve D, Figure 3. About 100 times the normal dosage of OxyC and HOM, showed a very small peak in the initial portion of the caffeine peak. However this situation does not exist even under the worst conditions of manufacturing. Thus solvents, OxyC, and HOM cause no interference in the analysis of caffeine, aspirin, hexobarbital, and phenacetin. The optimum conditions for the analysis of multicomponents present in the drug products are given in Table I. The six peaks along with their absorbance selectivity observed in Figure 4 are due to methanol, caffeine, aspirin, hexobarbital, phenacetin, and methylparaben (internal standard), respectively. The peaks are well resolved and it takes about half an hour for each chromatogram. For the analysis of samples not containing hexobarbital, benzoic acid is used as an internal standard instead of methylparaben, saving about five minutes for each analysis. Benzoic acid cannot be used in the presence of hexobarbital, since they both elute at the same time. OxyC and HOM cannot be determined by this system, but cause no interference ~~~~~
~~
Table 111. Quantitative Data-Area -
Figure 4. Liquid chromatogram of Percobarb sample showing methanol solvent, caffeine ( C ) , aspirin ( A ) , hexobarbital (HB), phenacetin (P), and methylparaben ( M P ) , respectively for the determination of other constituents, especially at the concentration levels in which they are present in the drug products of this study. The number of theoretical plates obtained by this 1meter long column varies from 190 to about 300 for all the peaks. The capacity factor which is a measure of sample retention varies from 0.25 to 9.0. The desired values for good resolution of the peaks from the mobile phase are 2 and above. The capacity factor for the caffeine peak from the solvent is not ideal but adequate for separation. The separation factor between the components varies from 1.5 to 4.5 which is very good. The resolution of the peaks, which is separation between the peaks divided by average peak width, varies from 1.5 to 2.0. Usually separations of 99% and above are obtained with a resolution of 1.25. Thus, the peaks are well resolved and base-line separations are possible. Quantitative Data. The concentrations of the active constituents present in the analgesics were determined by peak height as well as by the area of the peaks, Table 11. Disc integration was used to calculate the area. Peak height or area ratios were obtained in relation to the internal standard. The percentage accuracy for area method ranges from 0.1 to 3.3, while in the case of peak height it is 1.1 to 19.1. Thus, for quantitative work, the area method was adopted. Several samples having known amounts of active constituents were analyzed and the results are summarized in Table 111. Maximum accuracy of about 2% was obtained for all the compounds determined quantitatively. Thus this method competes well with other analytical techniques. Samples of Percodan and Percobarb were
A
Found, mg
Actual, mg
275.8 279.0 255.3 257.7 223.3 220.3
277.0 277.0 254.0 254.0 224.0 224.0
Method C
P
___ Accuracy,
Actual, mg
Accuracy
R
Found, mg
0.5 0.7 0.5 1.4 0.3 1.6
182.4 183.9 136.5 134.9 176.9 176.9
184.0 184.0 136.0 136.0 175.0 175.0
0.9 0.1 0.4 0.8 1.1 1.1
Table IV. Quantitative Data-Comparative
70
Actual mg
Accuracy,
38.9 39.4 37.7 36.8 34.9 35.1
39.3 39.3 37.0 37.0 35.3 35.3
1.0 0.1 1.8 0.5 1.2 0.5
mg
99.0 102.0
C, 29-35 mg
P, 144-176 mg
GC after
Percodan Percobarb
7%
Found,
Actual, mg
Accuracy,
100.0 100.0
1.0 2.0
70
Values
A, 202-246 mg Product
HB
Found mg
1,C
IR
LC
column sep.
LC
GC after column sep.
224 220
224 229
161 169
162 165
31.9 31.2
31.5 31.6
HB, 90-110 mg LC
GC after column sep.
... ioi:6
101.1
ANALYTICAL CHEMISTRY, VOL. 46, NO. 3, MARCH 1974
451
analyzed by liquid chromatography and the results obtained with gas chromatographic (for caffeine, phenacetin, and hexobarbital) and infrared (for aspirin) methods of analysis are compared in Table IV. As the maximum percentage relative standard deviation for all the compounds was about 2.8, the results obtained by these different analytical techniques compare favorably. The advantage of the liquid chromatographic method in eliminating several tedious separations involving Celite columns (as used in
gas chromatographic technique), saves considerable time for each analysis, and thus should be adopted for this investigation.
Received for review December 26, 1972. Accepted September 11, 1973. This paper was presented before the Division of Analytical Chemistry of the American Chemical Society a t the 164th National Meeting, September 1972, New York, N.Y.
Electron Capture Derivative for Determination of Nicotine in Sub-Picomole Quantities Lakshmanan Neelakantan and H. B. Kostenbauder College of Pharmacy, University of Kentucky, Lexington, Ky. 40506
Peak nicotine blood levels in humans immediately after smoking one cigarette may be of the order of 10 to 50 ng/ml of blood plasma, and these levels fall to one-half to one-fourth of the peak level in less than one hour. Gas chromatography, utilizing hydrogen or alkali flame ionization detectors, permits detection of nicotine in amounts of 0.2 ng and quantitation of 1 ng (1, 2). Of great value to investigators wishing to follow nicotine plasma and tissue 'levels following smoking by humans, or exposure of animals to smoke, would be an electron capture derivative which would permit quantitation of nicotine in biological fluids a t levels of 5 to 10 picograms. We wish to report a unique method for preparation of such a derivative. The procedure is based upon catalytic hydrogenation of nicotine (I) to yield N-methyl-4-(3'-piperidyl)-n-butylamine (octahydronicotine) (II), the two secondary amino functions of which may then be treated with a perfluoroacid anhydride to provide an electron capturing derivative. The di-pentafluoropropionyl derivative (111) of octahydronicotine can readily be quantitated in amounts corresponding to 0.03 picomole of nicotine.
pL CH3 H20
m
H
3
*2HCI
-2 HCI
m EXPERIMENTAL Chromatographic Conditions. The chromatographic system consisted of a Varian Aerograph, Model 2700, fitted with glass columns (length 2 meters, outside diameter 0.625 cm, inside diameter 0.20 cm) packed with chromosorb G, 100-120 mesh, coated with 1.25% OV 17 (Applied Science Laboratories, Inc., State (1) I . E. Burrows, P. J. Corp, G. C. Jackson, and 6. F. J . Page, Analyst (London),96,81 (1971). (2) P. F. Isaac and M. J. Rand, Nature (London),236,308(1972).
452
College, Pa.) and with both a hydrogen flame ionization and a 250-mCi tritium (EC) detector. The column was preconditioned for 36 hours a t 275 "C and was conditioned with Silyl 8 (Pierce Chemical Co., Rockford, Ill.) prior to use. For flame ionization detection carrier gas (N2) flow was 30 ml/min, hydrogen 30 ml/ min, air 300 ml/min, and injector port temperature was 245 "C, column 136 "C, and detector 200 "C. For electron capture detection, carrier gas (N2) flow was 30 ml/min, with injector port 245 "C, column 185 "C, and detector 215 "C. Reagents. Nicotine, 2HC1 (Baker Grade) was purchased from J . T. Baker Chemical Co.; platinum oxide, platinum black, and palladium on charcoal were purchased from Pfaltz and Bauer, Inc.; perfluoropropionic anhydride was purchased from Pierce Chemical Co.; n-heptane (pesticide grade) was purchased from Matheson Coleman and Bell; diethyl ether (anhydrous) was purchased from Fisher Scientific Co. Micro Catalytic Hydrogenation. In a 20-ml Kimax culture tube was placed 5 mg of 10% P d / C catalyst. A solution of 2 mg of nicotine dihydrochloride in 3 ml of water was carefully added, and the mixture was shaken and hydrogenated a t 20 psi for 4 hours a t room temperature. The charcoal was then filtered off on a sintered glass filter, washed with 2 ml of water, and the clear solution collected in a 15-ml centrifuge tube. The solution was adjusted to about pH 11 by addition of 5% NaOH, was extracted 3 times with 3 ml of diethyl ether, the ether solution was dried over Drierite, was filtered, and after concentration a sample injected into a gas chromatograph equipped with a FID showed a single peak with retention time of 4.75 min, (nicotine rt under these conditions is 3.0 min). Derivatization. A 2-mg sample of nicotine dihydrochloride was hydrogenated as described above, the octahydronicotine dihydrochloride solution was collected in a 15-ml centrifuge tube and evaporated to dryness at 55 "C under reduced pressure, and the residue dried at 70 "C under vacuum for 1 hour. To this tube was then added 0.4 ml of perfluoropropionic anhydride, the tube was sealed with a Teflon-lined screw cap and placed in an oven a t 70 "C for 2 hours. The excess anhydride was then removed by directing a stream of nitrogen into the tube. The residue was dissolved in 2 ml of heptane, which was then washed first with 2 ml of water (the acidic aqueous wash was saved) and then with 2 ml of 5% sodium bicarbonate solution. The heptane layer was then transferred to another 15-ml centrifuge tube and the solvent was evaporated with a stream of nitrogen. The residue was further dried at 40 "C under high vacuum (0.1 mm Hg) and redissolved in 1 ml of n-heptane. One microliter of this solution injected onto a gas chromatograph equipped with an EC detector produced a well-defined peak with retention time of approximately 9.5 minutes. The acidic aqueous wash solution was made basic with NaOH (pH E), extracted twice with 5-ml portions of diethyl ether, the combined ether phase was dried over Drierite, filtered, concentrated to 0.2 ml, and a 1-rl sample injected on a gas chromatograph equipped with a FID detector. No peak corresponding t o underivatized octahydronicotine was observable.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 3, MARCH 1974
