Clinical Applications of Infrared Spectroscopy. Analysis of Renal Tract

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Table Ill.

Organic Solvent Chloroform Ether

Partition Coefficient of Mephenesin and 3-(o-Tolyloxy)lactic Acid

Immiscible Phase Sodium hydroxide Citric acid Sodium hydroxide Citric acid

the theoretical distribution calculated from the partition coefficient (Table 111). There was no indication of a second material. The similarity of the distribution curves justifies the conclusion that the assay of 3-(o-tolyloxy)lactic acid in plasma is a measurement and does not include mephenesin. Riley (6) has identified 3-(4hydroxyo-toly1oxy)lactic acid in human urine following administration of mephenesin and reported that the compound has a n absorption peak at 290 mp. Following the administration of either mephenesin or 3-(o-tolyloxy)lactic acid to the animal, the assay of urine shows a small amount of material with a nonspecific absorption a t 290 mp. As only 70 to 80% of the 3-(o-tolyloxy)lactic acid administered to the dog intravenously was recovered in the urine after 4l/2 hours, this uncharacterized absorption may represent 3 - ( 4 hydroxy-o-toly1oxy)lactic acid.

Partition Coefficient’,K , f Std. Dev. 3-(o-Tolyloxy )lactic Mephenesin acid 0.206 f 0.017 0.380 & 0.010 4.155 & 0.093 1.873 f 0.084

1 . 9 9 4 . f 0.051 0 5.920 0.194

Recovery. T h e recovery of mephenesin added t o fresh plasma averaged 96.6 5.82yGin t h e range of 25 t o 200 y per ml. I n contrast, the recovery of mephenesin added to aged plasma averaged 106.5 f 4.35% within the same range. The recovery of 3-(0toly1oxy)lactic acid added to fresh plasma averaged 100.1 f 5.32%, again within the same range. The recovery from plasma containing less than 25 y per ml. of either compound ranged from 80 to l20%, averaging

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108.47G. Representative H u m a n Data. Blood

samples were obtained from 6 patients who had received 2 grams of mephenesin intravenously in a 12- t o 20-minute period. T h e plasma was assayed for mephenesin and 3-(o-tolyloxy)lactic acid b y the method described. Following the completion of injection, 3 to 7 y per ml. of mephenesin and 4.5 to 8 y per nil. of 3-(o-tolyloxy)lactic acid

n-as found. Thirty minutes after injection, mephenesin levels fell to 1.3 to 2.5 y per ml., while 3-(o-tolyloxy)lactic acid remained a t 5 to 10 y per ml. I n two cases in which data were available 60 minutes after injection, no mephenesin was detected; but 5 to 6 y per ml. of 3-(o-tolyloxy)lactic acid was present. These data indicate the applicability of this method to the determination, in human plasma, of mephenesin and its primary metabolite. LITERATURE CITED

(1) Friedel, R. il., Orchin, )I., “Ultra-

violet Spectra of Aromatic Compounds,” Kiley, Yen- York, 1951. ( 2 ) Graves, E. L., Elliott, T. J., Bradley, 11 ., Suture 162, 257 (1948). (3) IIellon, M. G., ANAL. CHEX 21, 3

(7) Stross, P.,’Stuckey, R. E., J . Pharm. nnd Pharmacol. 2, 549 (1950).

( 8 ) Titus, E., Ulick, S., Richardson, A. P., J . Pharrnacol. Exptl. Therap. 93, 129 (1948). (9) Kyngaarden, J. B., Koods, L. A., Seevers, hI. H., Proc. SOC.Exptl. Bzo2. Med. 6 6 , 256 (1947). (10) Yonden, IT. J., “Statistical Methods for Chemists,” Kiley, Yew York, 1951.

RECEII-EDfor review October 24, 1958. Accepted Ipril 2, 1959.

Clinical Applications of Infrared Spectroscopy Analysis of Renal Tract Calculi MILTON WEISSMAN, BERNARD KLEIN, and JUDITH BERKOWITZ Biochemistry and General Medical Research laboratories, Veterans Administration Hospital, Bronx 68, The exact composition of renal tract calculi is clinically important. Infrared absorption spectroscopy utilizing potassium bromide pressed-disk preparations is a newer approach to the analysis of inorganic and organic calculi. It has the advantages of speed and ease of sample preparation, permits a precise qualitative identification of both single components and mixtures and some semiquantitative estimations. Absorption spectra of the usually occurring calculi types and mixtures are compared with the spectra of pure compounds and prepared mixtures.

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knowledge of the composition of urinary tract calculi is clinically important to determine the RECISE

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ANALYTICAL CHEMISTRY

possible cause of stone formation and also to plan a program of medical management by dietary and other means to avoid recurrence. Clinical chemists usually must depend on qualitative analysis of these calculi because the specimens are most often fragments, on the order of a few milligrams. More exact and quantitative information can be obtained only infrequently when a sufficiently large stone becomes available. Recent examples of these tedious, time-consuming analyses are given by Leonard and Butt (9) and Nicholas (11). Inevitably, analysts turned to optical or other physical methods of analysis. An outstanding contribution was made by Prien and Frondel (12) \Tho examined about 6000 urinary tract calculi under a petrographic microscope with polar-

N. Y.

ized light, and with correlated x-ray diffraction patterns, related crystal structure to composition. Thus, for the first time definite structure and composition could be positively determined. However, these specialized instruments are rarely found in hospital laboratories. The availability of a n infrared recording spectrophotometer in this laboratory and information concerning the successful application of infrared spectroscopy to the identification of inorganic salts (IO), minerals, and rocks (6, 6),as n-ell as a recent publication on the infrared spectrophotometric analysis of dental enamel apatite (4) led to the application of this technique to the analysis of urinary tract calculi Beischer ( I ) briefly reported analyses of renal calculi by infrared spectropho-

tometry, whereby the samples were prepared by mulling in Sujol. This paper summarizes analyses of representative calculus types most frequently encountered in clinical practice by the potassium bromide pressed-disk technique which overcomes some of the major shortconlings of the Kujol or fluorolube mull. METHOD

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IO Representative spectra g

--Calcium oxalate monohydrate - - - Typical oxalate calculus

Infrared absorption spectra were obtained with a Perkin-Elmer hlodel 21 recording spectrophotometer, equipped Kith sodium chloride optics. The instrument was calibrated against a polystyrene film. For analysis, the calculi, where possible, were sectioned and if layers were visible, they were separated and analyzed separately. Otherwise, the calculus was crushed in a mullite mortar and ground as fine as possible. Disks were prepared by intimately mixing 1 to 2 mg. of finely ground material with 250 mg. of pure dry potassiuni bromide and compressing under vacuum for 2 to 3 minutes a t 20,000 t o 22,000 psi. Reference spectra n.ere prepared from the substances \J hich occur most frequently in renal calculi: calcium oxalate, hydroxy- and/or carbonatoapatites, oxalate-apatite mixtures, magnesium ammonia phosphate, mixtures of h I g S H 4 P 0 4 with oxalate, with apatite, or a combination of all three, urate, and cystine. RESULTS

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Representative spectra

-Synthetic carbonatoapatite - - - Typical apatite calculus

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7 ! J 9 II 13 Spectra of three natural hydroxyfluorapatites 1. 2. 3.

Rossie, N. Y. Bethel, Conn. Hingo Mine, Keystone, S. D.

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Calcium oxalate monohydrate (Figure 1) was precipitated by the procedure of Kolthoff and Sandell (8). The distinct \ m r e lengths of reference are the broad O H stretching frequency a t 2.85 to 3.0 microns, strong carboxyl absorption a t 6.10 and 7.5, the thvo weaker absorptions a t 10.5 and 11.25, and finally thp sharp absorption a t 12.70 microns. This agrees with published values (6). The spectrum given by a typical predominating oxalate calculus which contains on the order of 10 to 15% of apatite is also shown in Figure 1. A synthetic carbonatoapatite (Figure 2) was prepared according to Romo (16). The spectrum agrees with his findings of phosphate absorptions a t 9.66 and 9.14 microns, and carbonate absorptions a t 6.85, 7.03, and 11.4 microns. Small amounts of OH are still present in this preparation, as noted hja weak broad absorption a t 2.8 to 3.0 microns. Figure 2 also shows the spectrum given by a typical apatite calculus. Figure 3 sho\T s the spectra given 1 1 ~ three naturally occurring hydroxyfluorapatites. There is typical weak absorption a t 2.90 to 3.00 microns (OH), the pronounced phosphate absorption between 9.0 and 10.0 microns-namely. at 9.15, 9.35, and a small peak at 10.4 microns. VOL. 3 1 , NO. 8, AUGUST 1 9 5 9

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The spectrum giren by a mixture of equal parts by weight of a mixed calcium oxalate-carbonatoapatite appears to be a superposition of one upon the other (Figure 4). The spectrum of a mixed calculus containing calcium oxa!ate and carbonatoapatite is also shown. Magnesium ammonium phosphate (Figure 5) was prepared by precipitation as MgKH4P04.6Hz0 (8). A strong broad absorption a t 3.25 microns indicates the N E + [Colthup ($)I. Also characteristic is the very strong broad absorption between 9.0 and 10.0 microns given by Po4-3. Moderate absorption is noted at 6.05,7,15, and 11.35microns. This appears to be a composite of the spectrum given by i\Ig(HPO& and NH4H2P04 because the first exhibits the medium absorption at 6.07 and 11.35 microns and the latter the shoulder and medium absorption a t 7.1 microns. [Miller and Rilkins (IO)]. The spectrum of a triple phosphate calculus is also shown. A sharpening of the peak at 9.50 microns has occurred. The presence of a moderately absorbing band a t 13.0 microns indicates the presence of oxalate. Additional contamination is also present (see discussion). The spectrum given by equal parts by weight of mixed magnesium ammonium phosphate-calcium oxalate is shown in Figure 6. Again, it appears to be a n overlay of one upon the other. The absorption at 7.05 microns is reduced, and the broad 9- to 10-micron absorption is narrowed between 9.25 and 9.45 microns with a shoulder at 9.50 to 9.75 microns. The spectrum of a calculus that qualitatively showed the presence of such a mixture is also shown. Equal parts by weight of a magnesium ammonium phosphate-carbonatoapatite mixture gave the spectrum seen in Figure 7. The most distinguishing features are the pronounced absorption remaining at 6.10 microns and the broadening of the absorption from 6.90 to 7.10 microns with loss of definition seen in the spectra of the individual components of the mixture. Figure 7 also shows the spectrum given by a calculus which qualitatively showed a mixture of NH4+, Mgf2, Ca+2J PO 4 --8 ) and All the distinctive absorptions of a magnesium ammonium phosphateapatite-oxalate mixture are seen in Figure 8. Present are: Sharp oxalate peaks a t 6.10 and 7.50 shifted to 6.15 and 7.55 microns; the broad apatite (PO4+) absorption between 9.25 and 9.50 and the moderate absorption a t 11.40 microns and the less broad absorptions a t 2.90 to 3.10 and 6.80 to 7.10 microns seen in MgNH4P04. No calculus of this mixed type was found in this somewhat limited study. The potassium bromide disk infrared spectrum of pure crystalline uric acid

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(Figure 9) is siniilar to that published by Clark ( 2 ) and also shoim by Randall et al. ( I S ) . Their spectra were obtained as Nujol mulls. Figure 9 shows the spectrum of a urate calculus obtained by the pressed-disk technique. The infrared absorption spectrum of cystine (Figure 10) is identical to the one published by Koegel et al. (Y), by the pressed-disk technique, although over a 2- to %micron range only. h

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similar spectrum published by Randall et al. ( I S ) is based on a mulled preparation. A pure cystine stone spectrum as a potassium bromide disk preparation is a130 shonx. DISCUSSION

Infrared spectrophotometric analysis is valuable for the precise qualitative identification of inorganic and organic calculi. The composition of mixtures

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Figure 5.

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-Calcium oxalate-carbonatoapatite _ - _Mixed calculus containing calcium

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oxalate and carbonatoapatite

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-Magnesium ammonium phosphate

- - - Triple phosphate calculus

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-Magnesium ammonium phosphate-calcium oxalate mixture - - - Calculus containing magnesium ammonium phosphate-calcium

axalote mixture

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can be easily detected. K i t h additional experience and x i t h the preparation of templates of mixtures of knonn composition, quantitative estimations can be fairly exact. The latter phase was not the immediate purpose of this

Figure 7.

study. An additional advantage of this method is its rapidity. By the programming arrangcnient used in this study and by installation of the proper gears on the drum drive, a complete spectrum m-as obtained in 12 minutes on


-Magnesium

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ammonium phosphate-carbonatoapatite mixture Calculus composed of mixture of "a+, M g + 2 , Ca+%, P 0 4 - 3 ,

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ammonium phosphate-calcium

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Uric acid

- - - U r a t e calculus

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x 11 inch graph paper. No qual.tative scheme can compare. The potassium bromide pressed-disk method of sample preparation is more convenient than the preparation of S u j o l mulls for solid-state analyses. S o interfering absorptions b y the mulling medium are encountered. Recently some shift in steroid infrared spectra when the potassium bromide disk x i s used has been reported, due possibly to interaction with the salt (14). This has not been noted in the spectra reported here. The absorption peaks obtained n i t h Kujol mulls coincide exactly with values obtained by the potassium bromide disk technique. Beischer ( I ) estimated that about 1% apatite in oxalate calculi can be identified by the appearance of a slight dip at 9.5 microns. Similarly, in mixed stones containing lIgSHdP04, a shift to the longer P04-3 wave length appeared. He thereby claimed to be able to detect 5% apatite in a triple phosphate stone. Although a comparative shift to longer n-ave lengths was not noted in the pure hlgNH4PO4, in a typical triple phosphate calculus examined, a sharpening of the absorption a t 9.50 microns was noted with retention of the inflexions a t 10.20 and 10.40 microns, indicative of small amounts of apatite. T o these, one additional criterion for the demonstration of MgKH4P04 should be addedemphasis on the appearance of t h e shoulder a t 7.0 microns with a weaker absorption a t 7.25 microns. I n the mixed apatite-hIgNH4POicalculus (Figure ? ) , this is shifted to 6.85 and 7.07 microns. One conclusion became evident early in this study, even though a limited series had been examined and the major emphasis was on the identification and differentiation of important calculus types rather than exact qualitative or semiquantitative definition. Renal calculi were rarely entirely pure or completely homogenelous. -411 inorganic calculi examined were mixtures of a predominating primary constituent with widely varying amounts and types of a secondary constituent. For example, a predominating calcium oxalate stone could contain from 1 to 20% apatite and vice versa. Similar results were obtained in examination of binary triple phosphate calculi mixtures. I n this limited study, no ternary calculi could be distinguished, although trace qualitative reactions werr obtained for the presence of KH4+, Rig+*, Po4+, and oxalate. The chemical colorimetric or precipitation reactions which make up the typical qualitative analytical scheme are probably more sensitive than the infrared spectrophotometer for trace constituents. However, the discrepancy may be caused by variation in sampling. This is not a disadvantage, because only the major con8!,

VOL. 31, NO. 8, AUGUST 1959

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stituents of a calculus are clinically significant. Considering the urinary tract medium in which calculi are deposited where all the above ions are constantly in contact Yith the nidus of the calculus once formed, i t is inevitable that these ions become either precipitated or adsorbed on the predominant calculus type. There is no difficulty in interpreting the spectra of the two organic calculus types most frequently found. They are individually distinct and characteristic and incapable of confusion. I n this limited series, no mixed calculi n-ere found. At present, no conclusions can be drawn concerning frequency of occurrence of the major calculus types, because a small series was examined. Additional work is in progress.

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IP It Figure 10. Representative spectra

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ACKNOWLEDGMENT

The authors are indebted to John Herman, Bronx Hospital, for his gift of natural fluorapatites, and to Herbert Jaffe, Rockefeller Institute for Medical Research, for helpful discussions. LITERATURE CITED

(1) Beischer, D. E., J . Urol. 73, 653 (1955). (2) Clark, C. C., Ph.D. thesis, Columbia University, New Pork, N. Y., 1950; Publ. 1838, Univ. Microfilms, Ann Arbor. Mich. (3) Colthup, X. B., J . O p t . Sac. Am. 40, 397 (1950). (4) Fischer, R. B., Ring, C. E., -1s.4~. CHEM.29, 431 (1957).

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1-Cystine Pure cystine calculus

unt. J. >I., Turner, D . S.,Ibz‘d., 2 5 , (5!l% (1953). (6) Hunt, J. hf., Wisherd, &I.P., Boil ham. L. C., Ibid.,22, 1478 (1950). (7) Koegel, R. J., Greenstein, J . P..

(12) Prien, E. L., Frondel, C. J., ,J. Croi. 57, 949 (1947). (13) Randall, H. hl., Fowler, R. G.. Fuson. N.. Danol. J. R.. “Infrared Determination of Organic Structure,” pp. 121, 208, Van Sostrand, Self Tork, 1949. (14) Roberts, Glyn, Ax.4~. CHEX. 29, 911 (1957). (15) Romo, L. A., J . Am. Chent. SOC.76, 3924 f1954). . ,

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RECEIVEDfor review October 20, 1958. Accepted February 26, 1959. Division of A1nalytical Chemistry, 135th AIeeting, .1CS, Boston, Mass , April 1959.

Winitz, hlilton, Birnbaum, S. XI., McCallum, R. *4.,J . Am. Chern. SOC. 77, 5708 (1955). (8) Kolthoff, I. XI., Sandell, E. B., “Textbook of Quantitative Inorganic Analvsis.” rev. ed.. D. 362. 1Iaemillan. Sew“York, 1947. (9) Leonard, R. H., Butt, A. J.. C h . Chem. 1 , 241 (1955). (10) Miller, F. A,, Wilkins, C. H.. A h i ~ CHEM.24, 1253 (1952). (11) Nicholas, H. D., CZzn. C h ~ i4 , 261 (1958).

Diffuse Reflectance Spectrophotometry in the Ultraviolet Using Powdered Salts T. R. GRIFFITHS, K. A. K. LOTT, and M. C. R. SYMONS Department o f Chemistry, The University, Southampton, England ,The spectra of a variety of powders have been studied b y diffuse reflectance methods in the 200- to 1000-mp region. Compounds whose spectra in the solid state are known were used to check the reliability of the method and it is concluded that, with appropriate precautions, the resulting spectra are trustworthy at least down to 220 mp. However, surface effects are of considerable importance. To illustrate the potentiality of the technique, a variety of compounds whose spectra in the solid state have not been previously reported were studied. These included certain periodates, iodates, sulfur-oxy ions, sodium superoxide, and ozonide.

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visible and ultraviolet spectra of solutions has become a n important analytical tool. I n attempting to identify observed bands, i t is frequently of interest to study the role of the solvent and one relevant datum is the corresponding spectrum in the gas or solid phase. .\lso, for solids which are not soluble in readily available transparent solvents, a knowledge of the spectrum of the solid may lead directly to identification. By far the most satisfactory method for measuring the spectra of solids is to study single crystals in which the absorbing species has been diluted Kith an isomorphous material which is transparent in the spectral rcgion of EASUREMEST Of

interest. Hon ever, this procedure is always laborious and often impossible. I n alternative is to measure the diffuse reflectance spectra of the powdered solids, which have been diluted, if necessary, by mixing with another powder which has no absorption in the region to be studied. The purpose of this study was to examine this technique in an attempt to define the spectral region in which it may be used reliably with standard spectrophotometers, and especially to discover whether such spectra can justifiably be treated as characteristic of the bulk solid. Other aspects of this technique have been studied by various workers (9-12, 18, 23).

Clinical Applications of Infrared Spectroscopy. Analysis of Renal Tract

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