these base stocks. Using the paraffinic blend no-lead calibration for the olefinic, aromatic, and the high- and lowvapor-pressure gasolines resulted in mean errors of 0.0001, 0.0020, 0.0010, and 0.0009 g/USG, respectively. Application of the background correction resulted in corresponding mean errors of 0.0002, 0,0001, 0.0004, and 0.0007 g/USG. Therefore variations in base stock composition have only a small effect for no-lead gasolines. However, the small background correction is significant a t refineries where the acceptable lead level is usually 0.01 g/USG, and a t service stations when the lead level is near the specification of 0.05 g/USG maximum. Otherwise the background correction can be omitted for most no-lead gasoline monitoring applications. The effect of base stock composition is insignificant for low-lead and regular/premium gasolines because of the greater dilutions involved, and no background correction is therefore required. A simple dilution is, of course, more rapid than chemical pretreatment methods such as ASTM D3237-73 and is to be preferred for that reason. Including calibration and background correction, the analysis times are 30 min for one no-lead gasoline sample, and 2% hr for 20. T h e corresponding times for low-lead and regular/premium gasolines are 20 min and 2 hr. Flameless atomic absorption (9, 10) and more complex chemical pretreatment (11) methods have also been reported recently for the analysis of lead in gasoline. However, flame atomic absorption methods adequately cover the concentration range of current interest and are to be preferred because of their better precision and speed. In summary, the atomic absorption method described here is a simple dilution procedure that is not affected by differences in lead alkyls or gasoline base stock composition. Neither chemical pretreatment nor unusually careful attention to operating parameters is required. The method is rapid and has very good precision and accuracy.
ACKNOWLEDGMENT We thank J. E. Coffey, P. L. Hettinga, D. Kulawic, and W. M. Meston for experimental assistance, and T. Johnson for statistical discussions.
LITERATURE CITED (1) D. J. Trent, Atom. Absorp. News/., 4, 348 (1965). (2) N. Ouickert, A. Zdrojewski, and L. Dubois. Sci. Total Environ., 1, 309 (1972).
Table 11. Comparison of Results with O t h e r Methods a n d Exchange D a t a Lead Concentration, g / G a This Work
Colorimekic
0.008 0.0201 0.0270 0.0422
0.007 0.0200 0.02 64 0.0426
0.310 0.420 0.426 0.435 0.483 0.490 0.730 0.800
X-Ray
Fxchan e Aveb f
stan% dev.
0.31 0.426 0.423 0.435 0.461
* 0.05
0.431 0.433 0.433 0.485
1.32 1.84 1.90 1.91 2.45 2.49 2 -93 4.59
0.51 f 0.09 0.74 0.82 1.36 1.80 1.96 1.93 2.42 2.52 2.93 4.56
f
0.11
i 0.12 i 0.13
* 0.09 f
0.13
i 0.34
g/USG except for Canadian Cooperative Fuel Exchange data for which units are g/IG. Canadian cooperative fuel exchange results include standard deviation data. Other results are from the ASTM fuel exchange. Results were obtained in 1972 and 1973.
(3) Report EPS 1-AP-73-3, Air Pollution Control Directorate, Environmental Protection Service, Ottawa, Canada, March 1973. (4) R. A. Mostyn and A. F. Cunningharn. J. lnst. Petrol., 53, 101 (1967). (5) R. M. Dagnall and T. S . West, Talanta, 11, 1553 (1964). (6) M. Kashiki, S. Yarnazoe. and S . Oshirna. Anal. Chim. Acta, 53, 95 (1971). (7) Du Pont Petroleum Laboratory Test Method M112-71. E. i. Du Pont de Nernours & Co., Inc.. Wilrnington, DE 19898. (8) H. W. Wilson, Anal. Chem., 38, 920 (1966). (9) M. P. Bratzel and C. L. Chakrabarti, Anal. Chim. Acta, 61, 25 (1972). (10) M. Kashiki, S.Yarnazoe, N. Ikeda, and S . Oshirna. Anal. Lett., 7 ( l ) ,53 (1974). (11) K. Campbell and J. M. Palmer, J. lnst. Petrol., 58, 193 (1972).
RECEIVEDfor review November 18, 1974. Accepted February 27,1975.
Spect rophoto metric and Gas-Liquid Chromatographic Determination of Amitriptyline Horace E. Hamilton,’ Jack E. Wallace,’ and Kenneth Blum2 The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284
Amitriptyline, 10,11-dihydro-5H-5-(3-dimethylaminopropylidene)dibenzo(a,d]cycloheptene,and its monomethylamino analog nortriptyline are psychotherapeutic agents whose therapeutic value for the management of depresDepartment of Pathology. Department of Pharmacology.
sions has been well established. The ultraviolet absorption spectrum of amitriptyline is nonspecific and difficult to distinguish in biologic extracts from the background absorption contributed by normal biologic constituents. A number of spectrophotometric methods for the analysis of amitriptyline in biologic specimens have been described ( 1 - 5 ) ; however, these have generally lacked sufficient sensiANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
1139
Table I. Standard Curve of Amitriptyline Oxidation Product Ceric s u l f a t e u l f u r i c acid,
Alkaline permanganate,
Alkaline permanganate,
5 m l heptane
5 ml heptane
50 m l hexane
Amitriptyline
Absorbance,’
concn q.,, / m l
Absorbance‘
Absorbance/
concn, u g / m l
Absorbance
2 0.294 0.147 0.216 4 0.579 0.145 0.424 6 0.874 0.146 0.623 8 1.146 0.143 0.845 10 1.429 0.143 1.043 0.143 12 0.711 1.241 a Absorbance a t 250 nm. Values are means of triplicate determinations.
Absorbance/
concn, ug / m l
Absorbance
concn, e g / m l
0.108 0.106 0.104 0.106 0.104 0.103
0.144 0.271 0.411 0.556 0.691 0.825
0.072 0.068 0.068 0.070 0.069 0.069
Table 11. Urine Amitriptyline Determination by Semimicroprocedure Amitriptyline found
Concn,
iiglml
Absorbance“
*
@g/ml
*
8.0 0.842 0.010 7.45 0.09 0.414 i 0.018 3.66 i 0.16 4.0 0.201 i 0.015 1.78 i 0.13 2 .o 0.90 0.04 0.103 i 0.005 1.o 0.45 i 0.06 0.051 i 0.007 0.5 aAbsorbance at 250 nm, adjusted for mean blank of 0.026. Mean of triplicate determinations f standard deviation.
*
ow
‘\.
, 220
240
260
280
300
3iC
340
360
NANOMETERS
Figure 1. Ultraviolet absorption spectra of amitriptyline in water, 5 /*g/ml(-), and of amitriptyline oxidation product corresponding to a sample of equivalent concentration in heptane (- - -)
tivity and specificity, or have required extensive purification procedures. Wallace and Dah1 (6) described a spectrophotometric determination of amitriptyline based upon oxidation of the drug by alkaline permanganate to anthraquinone ( 7 ) , a product from the drug possessing a unique ultraviolet absorption spectrum and a high extinction coefficient. Although minor modifications of that procedure have been published ( 8 ) , it is considered by several investigators (9-11) to be the method of choice for the analysis of amitriptyline in urine. The current report describes a modification of the Wallace method that includes several innovative concepts not previously described. The present procedure provides enhanced sensitivity, increased stability of oxidizing reagent, a decreased volume of solvent requirement, and eliminates two steps, thus significantly reducing the analysis time. The drug as its oxidation product can be quantitatively determined by either ultraviolet spectrometry or gas-liquid chromatography. Both a semimicro and micro adaptation of the procedure are described. EXPERIMENTAL Reagents. A 5.5M sulfuric acid solution containing 25 mg of ceric sulfate per milliliter is prepared by adding 76 ml concentrated sulfuric acid, very slowly and with constant stirring, to a large beaker containing 6.25 g ceric sulfate and 174 ml water. The solution is stable for two months at room temperature. The n- hexane and n-heptane utilized are of spectroanalytical quality. Apparatus. Reflux condensers were mounted on a Flexaframe support. The semimicro determination utilizes a conventional Allihn condenser, the microdetermination utilizes a previously described “external cold finger” reflux condenser available from Kontes Glass Co., Vineland, N J (12). Heating mantles (Glas-Col, 1140
ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
270 watt, 500-ml capacity) were positioned upon magnetic stirrers beneath the condenser. Six reflux units were attached to a single Staco variable transformer which applied voltage to each of the heating mantles through the use of a CRC Multi-lectric Outlet. A similar outlet box applied line voltage to each of the six magnetic stirrers. A convenient reflux system may be achieved by mounting two sets of six reflux units each from a single support matrix, one set to the front and the other to the rear. The use of BB’s or shot pellets as a heat transfer media allows the heating mantles to be used with varying sizes of flasks, as required. Spectrophotometric measurements were performed on a Beckman ACTA CIII ratio-recording spectrophotometer equipped with a 3-cell Multi-Position mount with microcuvette holder. Quartz microcuvettes, 10 mm X 2 mm X 25 mm, of 0.4-ml capacity, were utilized for the microdeterminations. Quartz cuvettes, 10 mm X 10 mm X 45 mm, of 3.5-ml capacity, were utilized for the semimicro analyses. Gas chromatographic determinations were carried out on a Shimadzu GC-5A gas-liquid chromatograph equipped with dual flame ionization detectors, utilizing 3% OV-17, 100-120 mesh (2.0meter X 4 mm-i.d. glass column) at a column temperature of 245 “C and nitrogen flow rate of 40 ml/min. Procedure. Semimicrodetermination. Five ml of urine, adjusted to p H 11-12, are extracted into 25 ml n-hexane utilizing a twominute vigorous manual extraction in a small separatory funnel or 50-ml glass-stoppered tube. The hexane is transferred into a 50-ml glass-stoppered graduated cylinder, and the volume of recovered solvent measured. Ten ml of the ceric sulfate-sulfuric acid solution are added, and the cylinder is shaken vigorously for two t o three minutes. Nine ml of the acid extract are transferred to a 250-ml round bottom flask containing 5 ml of n-heptane and a magnetic stirring bar. Microdetermination. Two ml of serum, plasma, or urine are extracted into ten ml n- hexane and back-extracted into three ml of ceric sulfate-sulfuric acid solution as above. A measured quantity, i.e., 2.8 ml, of the acid extract and 1 ml n-heptane are pipetted into a 50-ml round bottom flask containing a magnetic stirring bar. The boiling flask is attached to the reflux condenser and secured into a heating mantle which is supported by the top surface of a magnetic stirrer. After the mixture has refluxed with vigorous stirring for 25 minutes (optimally achieved with 60 Vac applied to the heating mantles for the macroreflux system and 40 Vac for the microreflux system), the heating mantles are removed and the flasks allowed to cool. The flasks are removed and the heptane layer is removed and scanned spectrophotometrically over the range 350-
Table 111. P l a s m a Amitriptyline Determination by Microprocedure” Absorbance, tangent
Concn, u g l m l , added
Amitriptyline found,c (total absorbance)
techniqueb
T o t a l absorbance
Amitriptyline found,b* (tangent absorbance)
1.89 i 0.17 1.81 j: 0.23 0.133 j: 0.025 1.15 0.10 1.41 j: 0.10 0.105 j: ‘0.010 j : 0.83 i 0.15 0.79 0.04 0.060 j: 0.004 i 0.48 j: 0.08 0.41 0.01 j : 0.032 j: 0.004 0.30 j: 0.07 0.23 i 0.03 0.019 i 0.004 a Mean of triplicate determinations f standard deviation. Base line drawn between inflection point/minimums at approximately 235 and 258 nm. Adjusted for mean blank plasma absorbance of 0.038. Adjusted for mean blank plasma absorbance of 0.004. 2.0 1.5 1.o 0.5 0.25
0.307 0.202 0.156 0.106 0.081
j :
*
0.024 0.015 0.021 0.011 0.010
*
*
*
Table IV. Elimination P a t t e r n of Amitriptyline (and Nortriptyline) in Urine Following a Single O r a l Dose of 50 m g Amitriptyline HC1 Amitriptyline level, u g / m l a Subject
1C
2c
T o t a l amitriptyline excreted, u g b 3c
1
2
3
0.71 439 2054 77 0.38 2.91 A 0.58 224 368 268 0.28 0.90 B 1038 859 3.47 1.34 0.98 1457 C 635 557 0.99 1.54 1.57 536 D (female) a Mean of duplicate determinations. * Mean concentration adjusted for urine volume. Three collective 8-hr urine specimens per subject.
230 nm. Analysis at a single wavelength may be achieved by determining the absorption at 250 nm. For a standard, an aqueous amitriptyline solution was extracted and determined in a manner identical to that described for the biologic specimen. The n-heptane from the reflux flasks can be directly injected into the column of a gas-liquid chromatograph to provide a chromatographic analysis if desired.
R E S U L T S AND DISCUSSION The amitriptyline oxidation product and anthraquinone exhibited identical ultraviolet and infrared spectra as well as gas chromatographic retention data. The spectral and gas chromatographic characteristics of anthraquinone have been previously described (6, 7). Consequently, oxidation of amitriptyline with ceric sulfate yields anthraquinone, the product also obtained by the alkaline permanganate oxidation of the tricyclic compound (6). Derivatization to anthraquinone provides a markedly enhanced sensitivity and specificity for the determination of amitriptyline by ultraviolet spectrophotometry (Figure 1).A number of other oxidants (13-18) which afford high yields of products from various other drugs and have been utilized in other analytical methods were examined, but all provided insignificant amounts of anthraquinone. The ceric sulfate oxidation of amitriptyline resulted in a marked increase in sensitivity for the determination of that drug over that achieved with alkaline Permanganate. Table I presents data on the absorption of the oxidation product obtained by the Wallace method (6), a modification of the Wallace procedure in which 5 ml n-heptane was substituted for 50 ml n- hexane in the reflux step, and the macroprocedure described in this paper. It should be noted that the permanganate oxidation procedures for determining amitriptyline (6, 8) require 50 ml hexane for the final solvent as opposed to the proposed ceric sulfate oxidation method that requires only 5 ml heptane in the macromethod and 1 ml heptane in the micromethod. In addition to achieving a lower absorbance per amount of concentration, previously reported methods required ten to fifty times as much drug to achieve an equivalent final concentration of anthraquinone. To establish the efficiency and reproducibility of the proposed method, recovery studies were performed on urine containing 0.5 to 8 pg/ml amitriptyline and on plasma
containing 0.25 to 4 pglml. The urine assays were performed by utilization of the semimicro adaptation, and the plasma specimens were assayed by the microtechnique. These data are presented in Tables I1 and 111, respectively. The amitriptyline recovery over the concentration range examined was 9 1 f 6% for urine and 88 f 8% for plasma (mean of triplicate determinations, f standard deviation). For absorbances
