Fast separation and estimation of isomeric mononitrophenols

ANALYTICAL

BIOCHEMISTRY

35, ‘%4%Ct49 (1970)

Fast Separation

and

Estimation

of

lsomeric

Mononitrophenols T. GESSNER Department

AND

M. ACARA

of Biochemical Pharmacology, School of Pharmacy, of New Yorlc, Buffalo, New York 14214 Received

State University

June 9, 1969

During the course of our study on certain aspects of metabolism of nitrobenzene it became necessary to separate and estimate isomeric nitrophenols. Earlier work with these phenols relied on separation of o-nitrophenol from its isomers by means of steam distillation; the meta and para iso,mers were then approximately estimated after their partial chromatographic separation (1, 2). Park (3) determined a#ccurately the nitrophenols arising as metabolites of 14C-nitrobenzene, by carrying out isotopic dilutions, preparing derivatives of the nitrophenols, and recrystallizing these to constant count. Parihar et al. (4) performed an extensive study of 25 chromatographic systems and found that ptoluenesulfonates of mononitrophenols could be separated on acidic alumina with petroleum ether/ether solvent. Free nitrophenols separated from each other only partially in the chromatographic systems studied by Graham (5). Gasparic (6) achieved varying degrees of separation of the mononitrophenols by chromatography on formamidetreated paper; the best separation between the meta and para isomers was obtained in acetic acid/benzene solvent system (95:5) where approximate Rf’s were 0.89, 0.23, and 0.15 for ortho, meta, and para, respectively. Other investigators, while looking for metabolites of the insecticide parathion, addressed themselves to the problem of p-nitrophenol detection and estimation in biological materials (mainly urine). Thus, Gardocki and Hazelton (7) assayed p-nitrophenol after its reduction to p-aminophenol; the precedure resulted in approximately 57% recovery of the p-nitrophenol added to urine. Hladka and Hla,dky (8) pointed out the problem of interference by urine pigments in the determination of p-nitrophenol in urine extracts. They separated these interfering materials by chromatography on silica gel thin layer in acetone/n-hexane (2O:SO) solvent. In an earlier work, Lawford and Harvey (9) tried to minimize the quantities of interfering pigments 442

ISOMERIC

MONONITROPHENOLS

443

by extracting biological materials with amyl alcohol/diethyl ether/ petroleum ether mixture, they then estimated the extracted p-nitrophenol by indophenol reaction after its reduction to the corresponding aminophenol according to the method of Robinson et al. (1). Moye and Winefordner (10) developed a spectrophotofluorometric method for the estimation of p-nitrophenol in human urine after chromatography in ether on silica thin layers. They reported an average recovery of the order of 88% of the phenol added to urine. In view of the above reports it appeared to us that it should be possible to separate most conveniently the isomeric mononitrophenols, from each other and from any interfering pigments, by thin-layer chromatography, and then to estimate them by means of their ultraviolet absorption. In order to minimize losses of these volatile phenols from chromatograms and to prevent interference with the ultraviolet spectra of the eluates, we strived to use highly volatile nonaromatic solvents for the chromatography. Estimations of these phenols in biological materials present the additional problem of quantitative recoveries following a succession of extractions. The met’hod we describe minimizes losses due to manipulations. We also describe a modification in which a quantitative isotopic dilution is effected prior to the extractions, thus making the method accountable for any losses at any per cent recovery. MATERIALS

AND

METHODS

Ma teriab

The mononitrophenols used for this study were commercial products, recrystallized as necessary and chromatographically pure: ortho-, m.p. 4&5”, was from Eastman Organic Chemicals; meta-, m.p. 95-6”, and para-, m.p. 1123”, were from Fisher Scientific Company. The solvents, diethyl ether, ligroin (D, 0.70), petroleum ether (b.p. 38.253.9”), toluene, were Fisher certified reagents; 200 proof ethanol was from Publicker Industries, Inc. Silica gel G was from Brinkman Instruments, Inc. Liquid scintiIlation materials, 2,5-diphenyloxazole (PPO) and 1,4bis- [2- (4-methyl-5-phenyloxazolyl) ] benzene (dimethyl-POPOP) , were from Packard Instruments. 14C-p-Nitrophenol was from Tracer Lab, Waltham, Mass. Thin layers (250 ,A thick) were prepared with a Brinkman applicator by applying a slurry of the silica in water and air drying the plates for 24 hr at room temperature. Carbonate-treated silica thin layers were prepared in a similar manner from 30 gm of silica mixed with 60 ml of 0.1 M sodium carbohydrate.

444

GFJSSNER

AND

ACARA

Methods Detection of the nitrophenols was effected by exposing the chromatograms to ultraviolet light under ChromatoVue illuminator; the nitro compounds appeared as intensely quenching areas. Determination of o-, m-, and p-nitrophenols. Various quantities of nitrophenols were applied to silica thin layers so that each spot contained a mixture of the isomers. Chromatography was carried out in the solvent mixture diethyl ether/ligroin (5 :3) or diethyl ether/petroleum ether (5:3). The separated nitrophenols were quantitatively recovered by locating the area of silica adsorbing the isomers with the aid of ultraviolet light, carefully collecting the demarcated thin layer into test tubes, and eluting these with 2-5 ml of 1 N NaOH or with absolute ethanol for 30 min, after thorough mixing of the suspensions. The mixtures were centrifuged and spectrophotometric readings of the eluates taken at the stated wavelengths after appropriate dilutions with 1 N NaOH. The concentrations of the nitrophenols in the eluates were calculated by reference to previously constructed standard curves for optical density readings at wavelengths 280, 292, and 405 ~J.L for o-, m-, and p-nitrophenol, respectively, determined for standard solutions of the phenols in 1 N NaOH; the data are summarized in Table 1. Ultraviolet

TABLE 1 Absorption of Nitrophenol

Solutions

Nitrophenol Solution

o-

m-

P-

350 rnp 0.22

330 mp 0.17

305 rnp 0.85

280 mp 0.35

292 rn# 0.30

405 rnp 1.36

In ether: 10 fig/ml x max E km

In

1 N NaOH: 10 pg/ml h lTlRX

E ICrn

Extraction from biological materials. To determine the applicability of the analytical method for purposes of determination of mononitrophenoks in biological materials, recoveries of the nitrophenols added to mouse liver homogenates, or microsomal fractions, were tested. Conditions of hydrolysis of nitrophenol metabolites to the free nitrophenols were simulated as described below. (a) Procedure for extraction of nitrophenols from liver microsomal fraction: To a mixture containing 1.0 ml of liver microsomal suspension, 1.0 ml of soluble liver fraction and 8 ml of 0.2M phosphate buffer,

ISOMERIC

445

MONONITROPHENOLS

pH 7.4, was added 0.50 mg of a nitrophenol. The solution was made approximately 2 N with respect to hydrochloric acid by addition of the concentrated acid and sealed tube hydrolysis carried out for 4 hr at 100”. The nitrophenol was extracted from the hydrolysis mixture three times with 10 ml of diethyl ether and the ether layers were combined. The ultraviolet spectra of each ether extract were taken on a Beckman DB spectrophotometer and recovery of each nitrophenol was determined from optical density readings in diethyl ether by comparison with readings of standard solutions ; the data are summarized in Table 1. from liver homo(b) Procedure for extraction of W-p-nitrophenol genates: Livers were weighed and homogenized in 2 weights of 0.25 M sucrose, in a Potter-Elvejhem homogenizer and the homogenate was diluted 1:5 with the sucrose solution; 5 ml samples of this suspension were mixed in various ways with 0.1 ml of a solution containing 0.330 mg p-nitrophenol and 0.01 ml of a solution containing 0.12 &i; in 0.032 mg of 14C-p-nitrophenol; 1 ml of concentrated HCI was added; the tubes were sealed and heated at 100” for 4 hr. The p-nitrophenol was extracted 3 times with 3 ml of diethyl ether. The layers were separated by centrifugation after each extraction and the ether extracts combined. In order to arrive at a volume of solution that could be easily applied to a chromatogram the nitrophenol was reabsorbed from the ether extract into 0.1 ml of 1 N Na,OH ; this was then acidified with HCI and the nitrophenol then reextracted into 1 ml of diethyl ether. Determination phenol. Labeled

of the specific

activity

of the extracted

W-p-nitro-

p-nitrophenol contained in extracts was chromatographed and located as already described. The compound was eluted from silica gel with 2.5 ml of absolute ethanol or from carbonatetreated silica with 1 N NaOH. The concentration of the nitrophenol in the eluate was determined spectrophotometrically as already described. The radioactivity contained in the eluate was determined by scintillation counting described below. ScintdJation counting was effected in toluene scintillation fluid (1 liter of toluene contained 6 gm of PPO and 50 mg of dimethylPOPOP). The samples were counted to preset count to a standard error of 5% on the Nuclear-Chicago Unilux liquid scintillation counter, model 6850. Efficiency of the system, using a counting solution consisting of 1.0 ml of ethanol and 15.0 ml of scintillation fluid, was determined to be 78%. Aqueous eluates were counted with efficiency of 65% in scintillation mixtures containing 9.0 ml of scintillation fluid, 1 ml of toluene, 3.8 ml of absolute ethanol, and 6.2 ml of the aqueous sampIe.

446

GESSNER

RESULTS

AND

AND

ACARA

DISCUSSION

Separation and Recoveries from Chromatograms

It can be seen from Table 2 that the isomeric mononitrophenols can be separated adequately either on silica or on carbonate-treated silica, using a 5:3 mixture of diethyl ether with ligroin or petroleum ether. For quantitative work, carbonate-treated silica is preferred because the volatile phenols are better retained on it during chromatography and other manipulations. Losses from neutral or acidic silica can be substantial-for instance p-nitrophenol in quantities 10-100 & was TABLE

2 in Solvent Systems I and II I: diethyl ether/petroleum ether (5:3 v/v) II: diethyl ether/ligroin (5: 3 v/v)

RI Values of Isomeric Nitrophenols

Silica thin layer :

Carbonate treated

Solvent system:

I

II

I

II

0.88 0.75 0.40

0.66 0.40 0.24

0.94 0.79 0.67

0.77 0.50 0.40

o-nitrophenol m-nitrophenol p-nitrophenol

Untreated

recovered from unchromatographed silica plates to the extent of 951000/0, but after chromatography such recoveries ranged from 80 to 90%. Recoveries fom carbonate-treated silica were of the order of 100% (see Table 3) irrespective of manipulations, provided the compounds were eluted with sodium hydroxide (ethanol elutes only about half of the material present on carbonate-treated silica). o-Nitrophenol is especially volatile and for good recoveries had to be eluted promptly from any chromatograms. TABLE 3 Recoveries from Chromatograms on Carbonate-Treated Silica TLC Developed in Diethyl Ether/Petroleum Ether (5:3 v/v) y. recoveries Nitrophenol O-

mP-

5 108 106

Amounts applied to TLC, pg 10 25 50

75

110 108 97

93 103 -

96 106 98

93 104 97

ISOMERIC

Estimation

447

MONONITROPHENOLS

of p-Nitrophenol

by Isotopic

Dilution

Since the main purpose of our work was to develop a method for determination of isomeric mononitrophenols that might arise as in vitro metabolites, recoveries of the compounds added to liver homogenates or microsmal suspensions were tested. Recoveries were performed under the conditions that simulated those that would accompany a biological experiment and hydrolysis of nitrophenol conjugates was simulated, as described in “Materials and Methods.” We found that diethyl ether extraction of the above mixtures recovered the added nitrophenols with only small amounts of impurities, as was judged by the ultraviolet spectra of the ether extracts. The calculated recoveries ranged between 85 and 106%. It was desired to decrease the volumes of these original ether extracts (usually 9-30 ml) so that they could be easily applied to chromatograms. Concentration of the extracts by evaporation entailed heavy losses of nitrophenols, so the volumes were scaled down by extraction of the nitrophenols into a small volume of alkali, acidification of the alkaline layer, and re-extraction of the phenols into a small volume of diethy ether. Such re-extraction precedure resulted in about 88% recoveries. Since the method offered separated isomers of good purity it seemed reasonable to expect that, if combined with a quantitatively effected isotopic dilution, the method would offer an accurate and rapid way of quantitation of isomeric mononitrophenols. We t,ested t’his hypothesis by using one isomer, the p-nitrophenol-2,6-W. In order t.o test if the method is applicable irrespective of whether unlabeled p-nitrophenol is being estimated by addition of a known quantity of the labeled compound, or the labeled p-nitrophenol (such as may arise as a metabolite of radioactive nitrobenzene or appropriately labeled parathion) is being estimated by quantitative addition of the unlabeled compound, we tested the effect of various procedures of isotopic dilution. Experiments were carried out with each of the following mixing procedures: (A) premixed unlabeled and labeled p-nitrophenol, that is isotopically diluted p-nitrophenol, was added quantitatively to mouse liver homogenate, the mixture was mixed, acidified, and heated as described in “Materials and Methods”; (B) a q uantity of unlabeled p-nitrophenol was quickly mixed with a similar homogenate, and the labeled compound added quickly and mixed, then acid was added and the mixture heated; (C) the same as procedure (B) except that the ingredients were mixed for 5 min; (D) a known quantity of labeled p-nitrophenol was mixed in, then acid was mixed in quickly, then unlabeled p-nitrophenol was added immediately, and the mixture was well shaken

448

GESSNER

AND

ACARA

and heated. Recoveries, chromatography, and estimations of the specific activity were carried out as described under “Materials and Methods.” All of the above isotopic dilutions involved a 1:lO dilution of the labeled p-nitrophenol and should have yielded p-nitrophenol with the specific activity of 0.33 mCi/gm. The experimental values obtained are given in Table 4. There is good agreement between the values of TABLE 4 Specific Activity of W-p-Nitrophenol Recovered from Liver Homogenates Isotopic dilution procedure

Sp. act. in mCi/gm

A

B

C

D

0.32 0.34

0.32 0.32

0.35 0.32

0.31 0.27

specific activity arrived at, by method A, B, or C. Subsequent experiments using method C confirmed the validity of the method. In this manner p-nitrophenol contained in a liver homogenate mixture at the level of 5 pg/gm (5 ppm) could be easily and accurately determined. Method D gave understandably low values, illustrating that p-nitrophenol can be readily lost from acidified homogenates and showing therefore that isotopic dilution after acidification is an inappropriate procedure. In the context of this last, observation it is somewhat, surprising that Moye and Winefordner (10) did not observe substantial losses of p-nitrophenol when they subjected strongly acidified urine to heating under reflux. They reported recoveries of p-nitrophenol ranging from 80 to 97%. However, it is not clear from their description whether they added the phenol to posthydrolysis urine or before hydrolysis. SUMMARY

We have shown that o-, m-, and p-nitrophenols can be best separated from their mixtures by thin-layer chromatography on carbonate-treated silica gel with diethyl ether/petroleum ether (5:3), and that the compounds can be recovered from chromatograms and estimated by means of their ultraviolet absorption in 1 N NaOH solution. The compounds can be recovered from biological materials and quantitated, irrespective of percentage recoveries, by a method which utilizes isotopic dilut:on and chromatography. This latter procedure bypasses the usual laborious derivatizations, isolations, and repeated recrystallization of the compounds that usually accompany isotopic dilution quantitations. The method should be of particular interest in connection with toxicological studies

of parathion

when detection

and estimation

of p-nitrophenol

are

ISOMEXIC

449

MONONITROPHENOLS

sought. The current ready availability of 14C-p-nitrophenol the application of the method to such studies particularly

should make convenient.

ACKNOWLEDGMENTS This investigation was supported of Environmental Health Sciences, D. C. Thanks are due to Mrs. Cathy

by grant ES-00013 United States Public Kavai

for

technical

from the National Institute Health Service, Washington, assistance.

REFERENCES 1. ROBINSON, D., SMITH, J. N., AND WILLIAMS, R. T., Biochem. J. 50, 221 (1951). 2. ROBINSON, D., SMITX, J. N., AND WILLIAMS, R. T., Bioekem. J. 50, 228 (1951). 3. PARKE, D. V., B&hem. J. 62, 339 (1956). 4. PARIHAR, D. B., SHARMA, S. P., AND TEWARI, K. C., J. Chromutog. 24, 230 (1966). 5. GRAHAM, R. J. T., J. Chromatog. 33, 118 (1968). 6. GASPARIC, J., J. Chromatog. 13, 459 (1964). 7. GARDOCKI, J. F., AND HAZELTON, L. W., J. Am. Pharm. Assoc. 40, 491 (1951). 8. HLADKA, A., AND HLADKY, Z., J. Chromutog. 22, 457 (1966). 9. LAWFORD, D. J., AND HARVEY, D. G.. Analyst 78, 63 (1953). 10. MOYE, H. A., AND WINEINRDNER, J. D., .I. Agr. Food Chem. 13, 533 (1965).