Phosphorimetry as a Means of Chemical Analysis The Analysis of Aspirin in Blood Serum and Plasma J. D. WINEFORDNER and H. W. LATZ1 Department o f Chemistry, University o f Florida, Gainesville, Flu.
b Phosphorimetry is applied to the analysis of aspirin in blood serum and plasma. Aspirin in 0.4 mi. of serum or plasma is extracted into chloroform, the chloroform is evaporated, the residue is dissolved in E.P.A. solvent ( a mixture of ethyl ether, isopentane, and ethyl alcohol in (1 volume ratio of 5:5:2) which is placed in a quartz sample tube, the intensity of the phosphorescence emission at 410 m,u is measured using white light for excitation and the result is calculated in milligrams of aspirin per 100 ml. of serum or plasma using a working curve and a dilution factor. Aspirin in the concentration range of 1 to 100 mg. per 100 ml. of serum or plasma can be analyzed in less than 10 minutes with good accuracy and reproducibility. None of the constituents normally present in serum or plasma gave serious interference.
T
POSSIBILITY of qualitatively identifying organic compounds by phosphorescence spect ra was suggested by Lewis and Kasha ( 6 ) in 1944. Since that time, the phosphorescence spectra of hundreds of organic compounds ha\ e been tabulated ( 2 ); however, it was not until 1957 that Kiers, Britt, and Kentworth (6) published an evaluation of the method as a quantitative means of analysis and demonstrated the potential of the method by analyzing synthetic binary mixtures of compounds selected for their structural similarities. They showed that the mixtures could be resolved instrumentally by choice of excitation wavelength, by choice of emission wavelength, by choice of observation time after cut-off of excitation radiation, or by any combination of the above. Because of this verjatility, complex mixtures could be resolved instrumentally in a short time, nhereas analysis of the same mixture by many other techniques required time consuming physical separation of components before an accurate analysis could be performed. On the basis of the ciseellent work of Kiers, Britt, and Wentworth, a commercial spectrophosphorimeter ( I ) was introduced in 1960. As recently as Present address, Union Carbide Chemicalv C o , Division of Union Carbide C o p , Tarrytown, N. Y. HE
August 1962, Parker and Hatchard (10) published a review of the possibilities of using phosphorescence measurements in chemical analysis. McGlynn, Keely, and h'eely, (8)used phosphorimetry to analyze individually and in a mixture three organic molecules of petrochemical interest. Their results were compared with fluorimetric and absorptiometric methods. Up to this time, phosphorimetry has not been applied to the analytical determination of a trace organic compound(s) in actual samples, such as food, biological fluids, etc. The work reported in this paper was initiated to develop a practical application of the method, in this case the quantitative determination of aspirin in blood, and to explore further the fundamentals involved. Most molecules with conjugated ring systems usually give intense phosphorescence ( I S , 17'). Aspirin, with its single ring, is found to give extremely intense phosphorescence in the solvent E.P.A. (a mixture of ethyl ether, isopentane, and ethyl alcohol in a volume ratio of 5 . 5 : 2 ) I n the solxent E.P .I.,the phosphorescence of aspirin greatly exceeded the phosphorescence, for similar concentrations, of any of the constituents normally present in blood. An even further increase in selectivity resultcd Rhen a chloroform extraction is used to separate aspirin from srrum or plasma. By means of a single extraction of aspirin from serum or plasma using chloroform and the evaporation of the chloroform and dissolution of the aqpirin in E.P.X., it is possible to determine aspirin in 0 4 nil. of blood serum or p!asma in the concentration range of 1 to 100 mg. per 100 ml. of serum or plasma with good accuracy and precision and in less than 10 minutes. S o serious interference from any of the components normally present in blood serum is found. Because of the wide-spread uqe of salicyclic acid and acet)-lsalicylic acid, the determination of salicvlate in tissue, blood, and urine is of great importance, especially in toxicology and in conducting experiments wit11 othpr drugs (16). The determination of saliq late conccntration in serum or plasma by the colorimetric ferric salicylate complex method is common in clinical laboratories ( 1 4 ) . In the event, that aspirin is thr medication employed for treatment, the
sample is hydrolyzed to convert aspirin to salicylate ion. No direct method for aspirin is available so that it is necessary to perform two analyses, one before and one after hydrolysis, to arrive a t a value for the aspirin level in serum or plasma (16). This procedure introduces error when the aspirin concentration is low compared to that of salicylic acid, and it is time consuming. Also, the salicylate method is time sensitive (14) and requires a fairly large background correction when measuring salicylate levels below 10 mg. per 100 ml. of serum (7, 9). Because of the time problem and the inaccuracy of the salicylate method, it is difficult to study the exact mechanism of aspirin action when used as an analgesic, relaxant, and sedative. The method presented in this paper is direct and specific for aspirin and is rapid and accurak over a large concentration range. It would seem to be readily adaptable to toxicological use and to studies requiring the direct analysis of aspirin. 1
THEORY AND COMPARISON OF PHOSPHORMETRY WITH OTHER SPECTROMETRIC METHODS
The theoretical fundamentals and basic equations of phosphorescence have been discussed elsewhere (3, 11, 13, 17). Excellent reviews of the theoretical principles important to the analyst have been given by Kiers, Britt, and Wentworth (6) and by Parker and Hatchard (10). Phosphorimetry as a means of chemical analysis should have several advantages over the use of other spectrometric methods of analysis. I n phosphorimetry the sample is necessarily thermostated a t 77" K. by the use of liquid nitrogen, and so the measured results should not be susceptible to small temperature fluctuations which often cause errors in other spectrometric methods. The low temperature should also minimize quenching effects which are often appreciable in fluorescence studies done a t room temperature. The scattering of incident radiation and the background due to fluorescence impurities should be low in phosphorimetry due to the use of a rotating shutter. Phosphorimetric arialysis should be extremely selective due to the use of VOL 35, NO. 10, SEPTEMBER 1963
1517
a rotating shutter which allows phosphoroscopic resolution. In phosphorimetry, as well as in fluorimetry, the choice of excitation and emission conditions allows additional flexibility not present in most other methods. The weak phosphorescence emission of many molecules and the low background phosphorescence of many solvents should enhance this selectivity even more. I n fact, it should be poz.;ible to use white light for excitation in some case.; which should result in a higher spectral sensitivity. Phoaphorimetry hhould be more rapid than mojt spectrometric methods because of the flexibility in choice of experimental conditions. I n most phosphorimetric analyses, a single liquidliquid extraction of the solute into an organic solvent should be sufficient. In many caqes the sensitivity of analysis bhould be comparable to that obtained in fluorimetry. The amount of sample needed for analysis should often be extremely small because of the small sample volume needed for measurement and the good sensitivity of analysis. The accuracy and precision of phosphorimetry should he similar to, or slightly better than, that obtained in fluorimetry depending on the number and kind of sample preparation steps in each method. Several possible disadvantages of using phosphorimetry as a means of analysis should also be mentioned. The solvents available for phosphorimetric analysis are limited to those which are essentially “phosphorescence” pure and which can be cooled down to liquid nitrogen temperature to produce a clear rigid glass. Sources of contamination, such as stopcock grease and cleaning agents, the photodecomposition of solute by incident radiation, and the presence of the inner-filter effect result in errors in phosphorimetry as well as in many other spectrometric methods. The use of liquid nitrogen and small sample tubes which are somewhat tedious to fill. empty, and align do not comprise significant disadvantages when compared with most other spectrometric methods. EXPERIMENTAL
Apparatus. The instrumental setup is shown in Figure 1. The excitation source, a xenon lamp (XBO-150 W Osram Lamp. American Instrument Co., Inc., Silver Spring, Lid.), is operated by a 19.8-volt, 7.5-ampere power supply (Sola S o . 67-10-101, Sola Electric Co., Chicago, Ill.). The incident radiation is focused by a quartz lens on the entrance slit of the excitation monochromator (No. 33-86-25-05 UV-Visible hlonochromator, Bausch &- Lomb, Inc., Rochester 2 , X. Y.), or when using a filter or no monochromator or filter a t all, the radiation is focused by a quartz lens on the sample tube in the sample hous1518
ANALYTICAL CHEMISTRY
TOP V I E W
?
‘L FRONT V I E W
Figure 1. A. 6. C. D.
E. F. G.
H. 1. 1.
Instrumental setup
Xenon source Quartz lens Excitation monochromator Sample housing, Dewar assembly, and phosphoroscope can Emission monochromator Photomultiplier detector Recorder Photomultiplier photometer Cenlrifuge Filter-phoiomultiplier detector assembly
ing. The sample housing contains the revolving phosphoroscope can and the quartz Dewar flask into which is placed the sample tube. The phosphoroscope can had adjustable openings (0 to 5 cm. long X 1 cm. high), the centers of which are 180’ apart so that the sample is excited twice during each revolution. The emitted radiation from the sample is collected by the emission monochromator (No. 103420 UV-Visible Monochromator, Farrand Optical Co., Inc., New York 70, N. Y.) or a filter which is placed at 90” to the incident radiation. The dispersed radiation is detected by a photomultiplier tube, and the resulting signal is amplified by a high gain, high stability photomultiplier photometer (Model ph-200, Eldorado Electronics Co., Berkeley, Calif.). The amplified signal is displayed either on the meter on the photometer or on a 10-mv. recorder (Model G l l h Varian Strip Chart Recorder, T’arian Associates, Palo Alto, Calif.) which is connected to the output of the amplifier. The phosphoroscope can used is constructed by cutting two rectangular holes, 180’ apart, in an aluminum pipet can (KO. 22082, Will Corp., Atlanta 1, Ga.). The can is exactly centered and mounted on the revolving base of a centrifuge (5-15720, E. H. Sargent and Co., Chicago 30, Ill.) which is plugged into a variac that is used to adjust the speed. The centrifuge is shock mounted on the top of a small table, and the phosphoroscope can is placed through a hole in the instrument platform which
hold the monochromators, quartz lens mounting, sample housing and Dewar flask. The instrumental components are positioned as in Figure 1. Figure 2 is a detailed drawing of the cylindrical housing and sample housing cover. The housing cover holds the quartz Dewar flask tightly in the center of the phosphoroscope can. The quartz Dewar flask, shown in Figure 3, was specially constructed for this work (H. S. Martin and Son, Evanston, Ill.). The flask contains a n inner tube into which the sample tube is placed. The central tube is reinforced a t several points. The flask, wrapped with aluminum foil and then with black electrical tape, is centered into the housing cover with a rubber gasket. The housing cover fits snugly into the housing, a steel cylindrical tube, and is permanently attached to the instrument platform. As shown in Figure 3, a sample tube chuck is used to position and to hold tightly the sample tube in the center of the flask. d black cover cap is loosely placed over the top of the sample tube during a phosphorimetric analysis to prevent interference from room light. The xenon source is mounted on a hakelite base. An aluminum cylinder is placed over the lamp as a light shield, and the base is mounted as shown in Figure 1. A small hood must be placed over the xenon lamp because of the generation of toxic ozone by the high voltage starter. To avoid frost on the Dewar windows, it is necessary to allow a small stream
Figure 2. ing cover
Sample housing and hous-
of dry air to flow cmtinuously on the windows. To prevent light leakage from the excitation source of the emission monochromator entrance slit, a baffle made of a cylindrical flat black cardboard tube with entrance and exit slits (2 cm. X 0.5 cm.) is placed around the lower end of the Dewar flask and taped in place. I n addition, a strip of black felt was glued to the side of the sample housing so that it rubbed gently against the iihosphoroscope can as it turned. Two photomultiplier housings (Model 200C316, Eldorado Electronics Co., Berkeley 10, Calif.: , each containing an RCA 1P28 phJtomultiplier tube are used. One is mounted to the exit slit of the emission monochromator. The other is optically aligned a t an angle of 180' to the emission monochromator and equipped with a filter holder for use in filter phosphorimetry. To avoid light leakage, the spaces between the housing and the entrance slit of the emission nionochromator and the filter holder-phoi omultiplier system are sealed by use of black rubber tubes, By means of a gear connected to the chart drive rod of the recorder and a similar gear mounted to the wavelength drive of the emission monochromator, the wavelength drive was synchronized with the recorder drive. The quartz sampll? tubes (10 inches long, 2-mm. bore, 1-rnm. wall thickness, Thermal American Fused Quartz Co., Montville, 9.J.) are sealed at one end. The tubes are filled by means of a 3-ml. hypodermic syringe with a 3-inch hypodermic needle and are emptied by means clf a 12-inch hypodermic needle connllcted to a water aspirator. I n the aspirin ana ysis, no excitation monochromator or excitation filters are used, and so the quzrtz lens holder is positioned t o focus the radiation on a I-cm. segment of the quartz sample tube. The exit slit width of the
emission monochromator is fixed a t 1 mm. It is unnecessary to use the filterphotomultiplier setup because of the great sensitivity of the aspirin analysis. However, in some subsequent studies the filter-photomultiplier system will be used to enhance the sensitivity of analysis. After all components are firmly mounted and aligned, the emission monochromator is calibrated using a mercury arc and the phosphorescence spectrum of indole ( 4 ) . The above is a description of the experimental spectrophosphorimeter used in this study. The instrument is somewhat complex a t the expense of versatility. However, it should be stressed that the construction of a spectrophosphorimeter should not be deterrent to the use of phosphorimetric analysis. This is due to the availability of a commercial spectrophosphorimeter as well as a filter phosphorimeter which are marketed by the American Instrument Company ( I ) . Experimental Operation of Spectrophosphorimeter. OBT.4INING PHOSPHORESCENCE EMISSIOX SPECTRA. To obtain phosphorescence emission spectra uncorrected for instrumental variables, the following procedure is used. The xenon source is turned on and allowed to stabilize. This requires about 15 minutes. The centrifuge is turned on, and the variac is adjusted until the phosphoroscope can is turning a t the desired speed. I n all cases in this paper the variac mas adjuqted to 80, corresponding to a speed of 3600 r.p.m. For most analyses the speed is not critical. Only if suffirient decay occurs during the "off period" or if decay times are being measured is the speed critical. The determination of decay times by use of the phosphoroscope jpeed has been discussed by Kierb. Britt, and Wentworth ( 5 ) , and the effect of shutter speed on spectral measurements has been discussed by Parker and Hatchard (IO). The detection system is then turned on, and liquid nitrogen is slowly added t o the Dewar until it is cooled and boiling is at a minimum. The sample solution is introduced via a 3-ml. hypodermic syringe into a clean, dry sample tube. The tube is inserted into the sample tube holder and then slowly into the Dewar. The sample tube is turned until the alignment mark is properly positioned. The excitation monochromator is adjusted to the desired wavelength, or the desired excitation filter is used. The n-avelength knob of the emission monochromator is turned t o 200 mp. the chart drive chain is tightened, and the recorder pen is adjusted to the base line. =Ifter about 30 seconds of sample cooling, which result. in a rigid clear glass, the chart drive of the recorder is turned on, arid the phosphorrwence eniissioii spectra between 220 a i d 650 nip are recorded. The recorder ib turned off, and the proces- is repeated, if desired. This whole operation, including sample tube alignment, requires about 10 minutes. White (18) and Parker and Rees ( l a ) have described the method used t o correct emission
S A M R E TUBE
v:
,bTUBE
COVER
I
-1-
AND
CHUCK DEWAR C O V E R
t -
Figure 3. Sample tube holder and Dewar assembly
(and eucitation) spectra for instrumental factors. If. upon removal of the sample tube from the liquid nitrogen, it is observed that the sample is cracked or snowy, the above process should be repeated. OBTAINING PHOSPHORESCEXCE EXCITATION SPECTRA.No excitation spectra were recorded in this paper. However, if desired. they can be 0btainc.d in the manner described above for emission spectra except that the narelength setting of the emission-monochromator is fixed at the desired wavelength. The wavelength drive of Ihe excitation monochromator is varied linearly, and the output of the photomultiplier detector is recorded. PHOSPHORESCEXCE ISTEXSITI LIEASUREMEXTS OF STAXDARDS OR CNKXOWXS. Single intensity measurements are made as described in the section above on emirsion spectra except that the wavelength of the emission monochromator is set to the desired value (usually the navelength of m a w mum emission as found from the uncorrected emission spectra), The intensity of emission can then he read off of the photometer meter or the recorder. Procedure. CLEANING SAMPIL TUBES AXD HIPODERMICSYRISGE. All sample tubes and hypodermic syringes are initially cleaned ah follows. Warm (80' C.) concentrated HKOa (reagent grade) is uwd to rinse the tubei. The tubes are then rinsed with distilled nater and three tinies with acetone (spectral grade). Finally the tubes are rinsed with 1 ml. of E.P.A. (HartmanLeddon Co.. Philadelphia 43, Pa.). .ifter the cleaning procedure. only a rinse with the sample solution to be analyzed is necessary, ebpecially if the solutions all hnTe about the same VOL 35, NO. 10, SEPTEMBER 1963
0
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aspirin concentration or if the solutions are measured from the most dilute t o the most concentrated. ALIGNMENTOF SAMPLE TUBESIN PHOSPHORIMETER. To assure accurate alignment of sample tubes, each sample tube is marked with a scratch on one side. The sample tube holder also contains a scratch so that the two scratches are always aligned in any analysis. By means of using a standard 20 mg. of aspirin per 100 ml. of E.P.A. solution, it is possible t o place scratches on a number of tubes so that each tube when aligned will give the same reading for the same solution. This is more convenient than using a conversion factor to correct for tube differences. However, tubes are so easy t o clean that it is just as simple and more accurate to use the same tube for a series of determinations. Even when using the same tube for a series of determinations, it is always best to check alignment of the instrumental components by an occasional check of the phosphorescence intensity of the standard 20 mg. of aspirin per 100 ml. of E.P.A. solution. If misalignment is present, the Dewar assembly can be turned until the previous reading is obtained. OBTAIIiING ASPIRIN WORKIIiG CURVE. A working curve of phosphorescence intensity US. aspirin concentration in E.P.A. solvent was obtained over the concentration range of 0.01 t o 100 mg. of aspirin per 100 ml. of E.P.A. by measuring standard E.P.X. solutions at 410 mp by the procedure described above. mhite light was used for excitation. The intensities, plotted in the working curve, are corrected for the background emission of the E.P.A. solvent (background corresponds to only 0.03 mg. of aspirin per 100 ml. of E.P.A. and so is negligible for all E.P.A. solutions escept those containing less than 0.25 mg. of aspirin per 100 ml. of E.P.A.). ANALYSISOF ASPIRIN I N SERUMOR PLA~MA It. was impossible to add accurately known weights of aspirin t o serum or plasma because of the slow dissolution rate and its relatively rapid hydrolysis rate. For this reason a stock solution of 1.00 mg. of aspirin per ml. of water was freshly prepared, and the calculated volume of stock solution was pipetted into the serum or plasma immediately before analysis. Although this resulted in a significant dilution in the more concentrated samples, it was the only reliable means
of knowing the exact aspirin concentration in the Serum or plasma. The procedure for aspirin in serum or plasma is as follows. A 0.4-ml. aliquot of serum or plasma is added to a 10-ml. glass-stoppered graduated cylinder. To the cylinder, 0.1 ml. of concentrated HC1 and 7.5 ml. of chloroform are added. After 30 seconds of vigorous shaking of the cylinder, the layers are allowed to separate. A 1.00ml. aliquot of the chloroform layer is pipetted into a 10-ml. beaker. The chloroform is evaporated rapidly (3 minutes) by allowing a gentle stream of dry air to flow over the surface. To the residue in the beaker, 1.00 ml. of E.P.A. is added, and the solution is swirled. All of the E.P.A. solution is ,poured into a 3-ml. hypodermic sprmge and the plunger replaced. Half of the E.P.B. is used to rinse the sample tube, and the other half is placed in the sample tube for analysis. The sample tube is placed in the Dewar flask, and the relative intensity is measured using the experimental conditions described for obtaining the working curve. The relative intensity is corrected for background emission and is converted to an E.P.A. aspirin concentration from the working curve plot (Figure 5). This value can be converted to aspirin concentration in blood serum or plasma (units of milligram aspirin per 100 ml. of serum or plasma) by multiplying by the conversion factor 18.75-i.e.,
1 ml. CHC18
of deter-
KO.
minations 4 4 4 5
ing curve.
The background emission is obtained by analyzing a number of serum and plasma samples with no added aspirin by the same procedure as the samples containing aspirin. The background is taken as the average of these values, and for all serum or plasma samples containing more than 5 mg. of aspirin per 100 ml., a correction is not necessary. RESULTS
Saturated E.P.AI. solutions of the various components in blood serum and plasma were prepared, and only the iolutions of thiamine, riboflavin, tyro-
>
ci
1520
Milligrams of aspirin per 100 milliliters of s o l u t i o n Rel. std. Recovery, Present Found dev., Yo % 3.4 5.3
10.3 24.0 46.0 90.0
ANALYTICAL CHEMISTRY
)
mg. aspirin
Determination of Aspirin in Aqueous Solutions
2.5 5.0 10.0 25.0 50.0 100.0
ml. blood
100 ml. blood 100 i d . serum or plasma
Table 11. Table 1.
(
___-)-l (ml. E.P..:4)
ml. CHCL ml.-blood
sine, and tryptophan produced measurable phosphorescence. Cholesterol, bilirubin, thyroxine, uric acid, creatinine, glucose, urea, and vitamin C gave no phosphorescent emission. The amino acid, tryptophan, gave the highest intensity emission. A saturated E.P.A. qolution of tryptophan gave a relative intensity corresponding to an aspirin concentration of 0.2 mg. per 100 ml. of E.P.4. Standard freeze-dried serum samples (Warner-Chilcott, Morris Plains, N. J.) were extracted directly with E.P.A. and excited with white light which resulted in a broad emission with maximum intensity a t 440 mp, This corresponds closely to the emission of tryptophan. A series of five extractions on the same standard sample resulted in the same maximum with approximately the same intensity. Extraction of actual serum samples containing no added aspirin gal e the same maximum again with about the same intensity. When chloroform extractions of serum were evaporated, and the re-idue dissolved in E.P..\., the same maximum appeared but with an intensity corresponding to 0.03 mg. of aspirin per 100 ml. of E.P.A. This concentration in E.P.A. would result from an aspirin level in blood of about 0.5 mg. per 100 ml. of serum. Deproteinization of serum samples before chloroform extraction did not de-
10 8 6 8 8 I
KO.
determinations 4 5 5 5 5
96 106
103 96 92 90
of
Value read mg. from aspirin graph in 100 ml. E.P.A.
(
) X value read from work-
crease this background intensity. E.P.A. extractions of human albumin and human globulin (Xutritional Biochemicals Corp., Cleveland 28, Ohio) gave the same results as for standard or actual serum. The background intensity at 410 mp remained constant to within lo'% for all serum and plasma samples analyzed. Figure 4 shows the phosphorescence emission spectra of aspirin (uncorrected for instrumental factors) in E.P.A. at concentrations of 0.1 and 1.0 mg. per 100 ml. of E.P.X. In the same figure the phosphorescence background emissions of E.P.A. and of the chloroform
Determination of Aspirin in Citrated Plasma Containing Salicylic Acid"
Millierams of amirin per 100 Gilliliters o f plasma Rel. std. Recovery, Added Found dev., yo % 1.00 5.00 10.00 2 5 . 00 ' 50.100
on 10.10 2.1 on
_1.1_
8
9
