Jack E. Wallace, Horace E. Hamilton, Joel A. Riloff, and Kenneth Blum. A sensitive,highly specific spectrophotometric meth- od for quantitative determination.
CLIN.
CHEM.
20/2,
159-162
(1974)
Spectrophotometric Determination of Ethchlorvynol in Biologic Specimens Jack E. Wallace, Horace E. Hamilton, Joel A. Riloff, and Kenneth Blum
A sensitive, highly specific spectrophotometric method for quantitative determination of ethchlorvynol in biologic specimens is described. The procedure is based on conversion of ethchlorvynol to a product mixture consisting of two major components that strongly absorb ultraviolet light. Results can be obtained within 45 mm of receipt of a specimen. Additional Keyphrases: tions of ethychlorvynol analysis
spectrophotometry e concentrain blood e drug derivatization for
Ethchlorvynol (“Placidyl”, Abbott; 1 -chloro-3ethyl-1-penten-4-yn-3-ol) is a commonly prescribed nonbarbiturate sedative. Problems associated with
the use of this
drug
include
both
psychologic
physical dependence, as well as tolerance hol-potentiating side effects (1, 2). Several of fatal ethchlorvynol intoxication have ported (3-5). Because the drug is widely
has several associated hazards, nique for rapidly determining mens
is quite
and
and
alco-
instances been reused and
an analytical
tech-
it in biologic
speci-
valuable.
Gas-chromatographic
methods
for determination
of ethchlorvynol have been reviewed by Gibson and Wright (4). Many laboratories, however, do not possess this equipment. In addition, the high volatility of ethchlorvynol makes gas-liquid chromatographic (GLC) analysis of the drug often non-rewarding. Frings et al. (6) described a colorimetric method that was superior to the previously reported relatively nonspecific colorimetric assays for ethchlorvynol (7,
Department of Pathology, The University of Texas Medical School at San Antonio, San Antonio, Tex. 78829 (J.E.W. and H.E.H.); the Biochemistry Branch, USAF School of Aerospace Medicine,
Brooks
AFB,
macology, The University nio (K.B.). Received
Texas
(J.A.R.);
and
of Texas Medical
May 31, 1973; accepted
Oct.
Department
of Phar-
School at San Anto-
1, 1973.
Ethchlorvynol does not effectively absorb ultraviolet radiant energy, but Wallace et al. (9) demonstrated that it can be spectrophotometrically mea-
sured with high specificity and sensitivity by first oxidizing the drug to an ultraviolet-absorbing deny. ative. However, a disadvantage of the procedure was that the oxidation reaction required a steam distillation. This report describes a rapid, sensitive, and specific spectrophotometric determination of ethchlorvynol, a modification of the original method of Wallace et al., in which steam distillation is replaced by a simple reflux process.
Materials and Methods Apparatus
A variable
autotransformer
was used
in conjunc-
tion with a “multi-lectric” outlet to deliver 40 V ac to 270-W “Glas-Col” heating mantles. Water-cooled 40-cm condensers were attached to 250-ml roundbottom flasks that were positioned in the heating
mantles. Magnetic stirrers were positioned the heating mantles. We used an “Acta Cifi” Beckman spectrophotometer with 10-mm cells.
General
beneath
ratio-recording
Extraction
Two milliliters
of whole blood, serum,
or urine,
or
2 g of homogenized tissue are mixed vigorously for 5 mm with 20 ml of “Spectrograde” n-heptane. The extraction is not pH-dependent, and can be done effectively in a 25-ml stoppered graduated cylinder. After the heptane-specimen mixture has separated (centrifugation is not generally required) an aliquot of 10 to 15 ml of the heptane layer is refluxed with 10
ml of hydrochloricacid (1 mol/liter)for20 mm with continuous magnetic stirring. The temperature of the refluxingliquidsis100 ± 3 #{176}C Upon completion of the reflux and cooling (in an ice bath or cool tap CLINICAL
CHEMISTRY,
Vol. 20, No.2,1974
159
water) to near room temperature, the heptane layer (for non-tissue specimens) is scanned spectrophotometrically over the range 210-360 nm vs. a blank of
0.90
“Spectrograde” n-heptane. For measurements at a singlewavelength, the absorbance is measured at 247 n m. The heptane layer may be extracted (3 mm, man-
0.80
The aqueous
extract
containing
S S
S
I
S
S
t
I
t
0.70
ual) with an equal volume of semicarbazide (0.5 mol/liter) buffered at pH 3.5 with solid sodium acetate. The molarity of the sodium acetate is not critical and the semicarbazide solution is stable indefinitely when stored at room temperature in a brown
bottle.
S S S S
0.60
w U
z 0.50 0
C’)
the semicar-
0.40
bazone of the ethchlorvynol derivative is scanned against a semicarbazide blank, or the absorbance measured at 286 nm. To eliminate interference from ultraviolet-absorbing corn pounds normally present in
0.30 S
S
0.20
tissueand tissueextracts, the semicarbazidereaction isrequiredforthe analysisoftissuespecimens.
0.10
S
S
Results S
Oxidation of ethchlorvynol results in a product(s) having a well-defined absorption curve (Amax, 247 mu) with an absorbance/concentration ratio of 0.065. Conversion of the oxidation product(s) to the semicarbazone derivative yields a sharp curve with the maximum at 286 nrn and an absorbance/concentration ratio of 0.093 (Figure 1). A linear relationship exists between the concentration of the drug, the absorbance of the oxidation product(s) and the formation of the semicarbazone (Figure 2). The linearity existsover the range of ethchlorvynol concentrations usually encountered in biologic specimens (3-5). If an exceptionally high absorbance is obtained, it is measured on a dilution of the final solution. Such an application is practical because the oxidation of ethchlorvynol is nearly stoichiometric over a wide concentration range (Figure 2), a marked advantage over available colorimetric procedures that require a new determination with smaller volume of specimen ifthe initial analysis shows too great an absorbance. Semicarbazone blanks forurine,serum, or tissue homogenates should have an absorbance of less than 0.03. Heptane blanks for tissue extracts are often high and variable, necessitating formation of the semicarbazone derivative. The carbonyl and the semicarbazone blanks obtained from oxalated whole blood are equivalent to those obtained with serum. Recovery. We determined the percentage recovery of ethchlorvynol from biologic specimens assayed by the present method. Aqueous solutions (containing 2 ml of ethanol per 100 ml) of ethchlorvynol standards were added to urine and serum to provide concentrations ranging from 1 to 15 mg/100 ml, and to homogenized rat tissues (liver, lung, brain, kidney, and fat) to provide a concentration of 10 mg/100 ml. Determinations were made on 2-ml amounts of urine or serum, or homogenate aliquots equivalent to 2 g of tissue. Recoveries are summarized in Table 1. Double extraction of serum, with subsequent combining of the hexane extracts, provided recoveries equiva160
CLINICAL
CHEMISTRY,
Vol. 20, No.2,
1974
220
240
260
280
300
320
340
360
NANOMETERS
Fig. 1. Ultraviolet absorption spectra of total ethchlorvynol products bazone of the carbonyl derivative --,
-
a 10 sg/mI solution
-
-
of ethchlorvynol and of the semicareach derived from -,
of ethchlorvynol
2
to uJ
o z m 0,
m
08-
06
04
0.2
2
4
I
I
I
I
6
8
0
2
ug/mI
Fig.
2.
Standard
-0-,
product,
and
the
-
14
16
CONCENTRATION
curve for ethchlorvynol reaction product, semicarbazone of the ethchiorvynol
-#{149}-
lent to that achieved for the analysis of urine after a single extraction. Tissue recoveries were lower and more variable than those of urine and serum. Interference. A number of drugs were investigated for possible interference. None was found to interfere significantly
(A
