Micromethod for Measuring Hexachiorophene in ... - Clinical Chemistry

1 downloads 0 Views 732KB Size Report
fore analysis, diacetyl derivatives of hexachlorophene and dichlorophene were formed by procedures similar to those used by others (1-5, 7). Materials and ...
CLIN.CHEM. 23/6, 944-947 (1977)

Micromethod for Measuring Hexachiorophene in Whole Blood by Gas-Liquid Chromatography W. Edwin Dodson,1 Eileen E. Tyrala,2 and Richard E. Hlilman3

Ill. 60068), equipped with a Varian Aerograph 63Ni We describe a micromethod for measuring hexachiorophene by use of gas-liquid chromatography with a #{176}3N1electron-capture detector, was used. This detector has electron capture detector. The procedure requires 100 il a linear range of only 50-fold and the detector responses of blood for extractions of hexachiorophene, and dichloto hexachlorophene and dichlorophene differ because rophene is addedasan internal standard. CV is 3.4% over of their different molecular contents of chlorine, so it the concentration range of 500 to 1300 g of hexachlowas necessary to determine the linearity range for each rophene per liter of whole blood. This procedure permits compound independently before constructing standard repeated measurements of hexachiorophene In newborns curves and establishing procedures for extraction of who are being washed with soap containing hexachioroblood samples. The gas chromatograph was operated phene. AdditIonal K.yphrases dichk,tvphene#{149}M detector pediatric chemistry newborns .

eleclmn capture

.

We describe a micromethod for measurement of hexachiorophene, 2,2’-methylenebis(3,4,6-trichlorophenol), by gas-liquid chromatography, which permits its repeated measurement in blood samples from babies who are being washed with soaps that contain it. Previous reports indicated that electron-capture detectors-including 63Ni (1-4), tritium foil (5-7), and helium discharge (8)-are adequately sensitive for these measurements, and we selected a 63Ni electron-capture detector for these studies. Whereas previous methods require from 1 to 3 ml of blood for extraction of hexachlorophene, volumes that preclude repeated measurements in small full-term and premature infants, our procedure is based on extracting hexachiorophene from 100 l of whole blood. To improve the precision of these measurements, dichlorophene [2,2’-methylenebis(4chlorophenol) was added as an internal standard. Before analysis, diacetyl derivatives of hexachlorophene and dichlorophene were formed by procedures similar to those used by others (1-5, 7).

Reagents

Materials and Methods Equipment

Gas chromatography. A Varian Model 2100 gasliquid chromatograph (Varian Instruments, Park Ridge, ‘The

Edward

partment Washington

Mallinckrodt

of Neurology University

Department

and Neurological

of Pediatrics

and De-

Surgery (Neurology),

School of Medicine, and the Divisions of Neurology,’ Neonatology,2 and Medical Genetics,3 St. Louis Children’s Hospital, St. Louis, Mo. 63110. Received Aug. 10, 1976; accepted Mar. 16, 1977. 944

CLINICAL

with injector, oven, and detector temperatures of 275, 220, and 300 #{176}C, respectively, and with an N2 carrier gas flow-rate of 35 mi/mm. Acid-washed siliconized columns, 0.4 to 0.5 m long, were packed with 3% 0V225 on 80/100 Supelcoport (Supelco, Inc., Bellefonte, Pa. 10823), with no glass wool at the injector end of the column. Mass spectrometry. We confirmed the formation of the diacetyl derivatives of hexachiorophene and dichlorophene by using a Model 3200 computer-operated quadrapole mass spectrometer (Finnigan Corp., Sunnyvale, Calif. 94086) interfaced to a gas-liquid chromatograph. The conditions of operation for obtaining electron impact mass spectra included an ionizing voltage of 70 eV, with other conditions similar to those used for gas chromatography. Mass spectra were also obtained with a computer-operated LKB-9000 mass spectrometer (LKB Instruments, Chicago, Ill. 60634) with gas-liquid chromatograph interface, operated at a source temperature of 270 #{176}C with an ionizing voltage of 70 eV. All glassware and Teflon cap liners were rinsed in ethyl acetate before use, to avoid contamination.

CHEMISTRY,Vol. 23, No. 6, 1977

USP hexachlorophene (Givaudan Corp., Clifton, N.J. 07014) was donated by Dr. Jean Holowach and dichlorophene was obtained from Aldrich Chemical Co., Milwaukee, Wis. 53233. These compounds were found by gas-liquid chromatography to be of satisfactory purity and did not require further purification. Acetic anhydride and pyridine (Fisher Chemical Co., St. Louis, Mo. 63132) were redistilled before use. “Pesticide Grade” ethyl acetate, N-hexane, acetone, and methanol

00 III

V

z 4

z 4 ‘U

>

IL1. I. 20

ti

‘56

J 160

150

I

ii 260 rn/C

ii

250

IIL

-

HCP ‘U

U

z

4 0

z

4 U.’

>

Fig. 1. Computer-plotted 70 eV electron Impact mass spectra of diacet)

from Fisher

Chemical

Co. were used without

further

purification. Preparation

of Standard Curves

Standard curves were prepared by use of stock solutions of hexachlorophene and dichiorophene in methanol, the solute being transferred to whole blood as follows: 500 ng of dichiorophene and from 5 to 250 ng of hexachlorophene were pipetted into screw-cap test tubes with Teflon cap liners. After the methanol was evaporated with nitrogen at room temperature, 0.1 ml of heparinized blood was added to each tube, and the samples were mixed in a vortex-type mixer. Preliminary work indicated that dichiorophene was extracted well by hexane but hexachiorophene was extracted more efficiently by ethyl acetate. Thus the blood samples were first extracted with 6 ml of hexane, then with 6 ml of ethyl acetate. In each case the samples were mixed for 30 s with a vortex-type mixer, centrifuged (5 mm, 1000 rpm), the organic layers removed with a transfer pipette, pooled, and evaporated under nitrogen in 2.5-ml screwcap reaction vials. Acetyl derivatives were prepared by reacting the residue with 100 Ml of an equivolume mixture of pyridine and acetic anhydride for 30 mm at 80#{176}C on a heating block, with the reaction vials capped (Teflon cap liners). The acetic anhydride and pyridine mixture was evaporated under nitrogen, the residue was redissolved in 0.2 ml of ethyl acetate, and 0.5 to 2 Mlof this solution was injected into the gas chromatograph. Procedure

for Patients’ Samples

Hexachlorophene was extracted from whole-blood samples from patients by a slightly modified procedure. Dichlorophene, 500 ng, in methanol was pipetted into 15-ml screw-cap tubes and the methanol removed in a

stream of nitrogen, at room temperature. These tubes containing the residue of dichlorophene were stored at room temperature before use; we found the dichiorophene to be stable for at least two weeks. The samples from patients were collected by heel puncture after cleansing the skin three times with acetone. Just before sample collection, 1 ml of hexane was added to each tube. A iOO-Ml volume of blood was collected in a disposable pipette, transferred to the screw-cap test tube, and the pipette rinsed with hexane. The subsequent extraction and derivatization of these samples were identical to those used in preparing standards in whole blood. Although the acetyl derivatives of hexachlorophene and dichlorophene were what was actually measured, all results are expressed in terms of hexachlorophene and dichiorophene. The concentrations of hexachlorophene in blood samples from patients were calculated from a standard curve prepared by plotting the ratios of peak heights of hexachlorophene to dichlorophene vs. the equivalent hexachlorophene concentration in the standards.

Results The combined gas-liquid chromatography and mass spectrometry of standards indicated that the diacetyl derivatives of hexachlorophene and dichlorophene were formed. No underivatized compound was detected. Mass spectra of these compounds are shown in Figure 1. A molecular ion for diacetyldichlorophene at mle 352 was present in low abundance. The base peak of m/e 128 and the next most abundant peak at m/e 140 had neighboring isotopic clusters indicating one chlorine, consistent with molecular formation of C6H5OCP and C7H5OC1+, respectively, whereas the third most abundant peak, at m/e 268, had an isotopic cluster indicating CLINICAL CHEMISTRY,

Vol. 23, No. 6, 1977

945

0

z

‘-I I.

w U I

I. 4 U 0.

O

2

6

1

lh

, TIME

Fig. 2. Left: a chromatogram of a mixture of diacetyldichiorophene (1) and diacetylhexachlorophene (2) extracted from supplemented whole blood. Right: chromatogramobtainedafter similarly processing an equal volume of the same blood without added hexachlorophene and dichlorophene Attenuator setting for both: 8 X

two chlorines, consistent with C13H1002C12+. A molecular ion of m/e 488 with appropriate isotopic cluster was observed for diacetyl hexachlorophene in low abundance. The isotopic patterns associated with fragmentation ions of mle 196, 404, and 446 indicated three, four, and six chlorine atoms, respectively (9). Short columns packed with 3% 0V225 gave the most satisfactory chromatographic results. Although the retention times for these compounds varied from one column to another, they were consistent for a given column. With a column 0.5 m long, a flow rate of 35 ml/min, and an oven temperature of 220 #{176}C, retention times for diacetyldichlorophene and diacetylhexachlorophene were 3 and 9.5 mm, respectively (Figure 2). Increasing the column temperature to 235 #{176}C decreased the retention times to 1.8 and 4 mm, respectively. It was necessary to eliminate the customary inlet plug of glass wool because some of the sample was deposited on it and eluted off with subsequent injections of solvent, producing “ghost” peaks. Whereas previous workers have found it useful to add silica gel to absorb contaminants which prolong the solvent front (1, 10), we found this unnecessary. Preliminary work indicated that our 63Ni electron capture detector had a linearity range for hexachlorophene and dichlorophene from 0.02 to 1.0 ng and 0.25 to 10 ng injected when the chromatographic conditions described above were used. For practical purposes, we found that a recorder response to either compound of a peak height greater than 120 mm at an attenuator setting of 8 X 10_b indicated the upper range of linearity and the sample was diluted and injected again. Reproducibility was good at different attenuator settings. It was necessary to standardize techniques of sample injection carefully because of the different sensitivity of the detector to hexachlorophene and dichlorophene and because of the different propensity of these corn946

CLINICAL CHEMISTRY,

Vol. 23,

MG/L

HEXACHLOROPHENE

Fig. 3. Standard curve prepared from data obtained on extraction of drug-supplemented whole-blood standards

10_b

No. 8, 1977

pounds to evaporate from the needle within the injection port. Varying the time the needle was left in the injection port from 2 to 20s produced a CV of 12.5% in peak-height ratios for repeated injections of the same sample. To avoid this loss of precision it was necessary either to make rapid injections with prompt withdrawal of the needle or to draw up solvent into the syringe before aspirating the sample, so that the needle was flushed with solvent after the sample was injected. With either of these methods, the CV for the peak-height ratios for five injections of the same sample was 1.21%. Whereas standard curves of the peak-height ratios of hexachlorophene to dichlorophene prepared in organic solvents gave linear plots with intercepts of zero, preparation of standard curves for which blood from personnel in the laboratory was used sometimes gave intercepts differing from zero (Figure 3). This was usually due to the occurrence of small peaks equivalent to 20 to 50 tg of hexachlorophene per liter. Thus it was helpful to include an unsupplemented blood sample from the same donor in preparing standard curves in whole blood, to correct for this background error. Three samples of cord-blood that were selected without conscious bias showed no peaks coincidental with those for hexachlorophene or the internal standard. For five analyses of each of three different samples the CV was 3.4%. Extraction efficiency was determined by the use of 15 supplemented standards, which were compared to identical amounts of hexachlorophene and dichlorophene pipetted directly into reaction vials. The average percentage extraction for both was 97% over a concentration range of 0.5 to 1.5 mg of hexachiorophene per liter, and at a dichlorophene concentration of 5 mg/liter, the concentration used as internal standard in patients’ samples. Extraction of the samples with either ethyl acetate alone or ethyl acetate followed by hexane decreased the percentage extraction of the in-

-

-J

800

/

600 0

-J

I 0

200 U

I

p

0

4

p

8

I

2

6

I

20

I

24

HOURS

Fig. 4. Plot of concentrations of hexachlorophene In blood vs. time for a 1250-g,three-day-old infant after the second bath with soap containing hexachlorophene (15 g/kg) The arrow indicates the time of the second bath

ternal standard,

but standard

neurotoxicity. This lack of information probably is a result of technical limitations in performing repeated measurements of hexachiorophene concentrations in newborns, because previous procedures have required large blood samples for drug extraction. Micro-scale methods such as ours permit the repeated measurement of hexachiorophene concentration in a newborn when its use is clinically warranted to curtail serious nosocomial infections. Figure 4 shows an example of the application of this method to repeatedly measure hexachlorophene in a three-day-old premature newborn weighing 1250 g receiving his second bath with soap containing 1.5% hexachlorophene. Future measurements of hexachlorophene under circumstances such as these, combined with careful clinical and pathological studies, can help in the development of guidelines for use of hexachlorophene.

curves were still linear.

Discussion Previous studies indicated that hexachlorophene can be measured by gas-liquid chromatography with use of a variety of derivatives and column packings (1-8, 10). Although methyl esters formed with diazomethane (1, 6, 10) and trimethylsilyl esters (1,8) have been reported, acetyl esters are reliably, safely, and easily produced. Whereas previous studies indicated satisfactory chromatography of hexachlorophene with several different column packings, our incorporation of dichlorophene as an internal standard restricts the selection of column packings. Our experience with 3% SE-30 and 3% OV-17 indicated relatively wide chromatographic separation of diacetyl hexachlorophene and diacetyldichiorophene with the SE-30 and partial deposition of hexachlorophene with the production of ghost peaks after solvent injections with OV-17. 3% 0V25 gave satisfactory separation of these compounds, and retention times were short enough that many samples can be processed. Previous reports have indicated that citrate (6) or phosphate (5) buffers improve the extraction of hexachlorophene by ethyl acetate. We found the use of buffers unnecessary, probably because of the large ratio of organic solvent to aqueous phase (60/1) we used. Because electron capture detectors are so sensitive to contamination, any simplification of the extraction that decreases the chance for contamination is desirable. Epidemiological studies have indicated possible neurotoxic effects in newborns washed with soaps containing hexachlorophene and have led to restrictions of its use (11). These studies have not correlated pathological findings with concentrations of hexachlorophene in blood or tissues from babies, and it is not known what threshold concentration is associated with

This work was supported by grants from the National Foundation-March of Dimes, the Ranken Jordan Trust, and NIH Mass Spectrometry Resource Grant RR-00954. Technical assistance was provided by Gail Reisenberg and John Allen. W.E.D. is the recipient of Nil-lAcademic Career Development Awardl-K07-NS111074.R.E.H. is the recipient of NIH Research Career Development Award 1K04-AM0008O.

References 1. Ulsamer, A.G., The determination of hexachiorophene in mammalian tissues by gas-liquid chromatography. J. Assoc. Off ic. Anal. Chem. 55, 1295 (1972). 2. Ulsamer, A.G., and Marzulli, FN, Hexachiorophene concentrations in blood associated with the use of products containing hexachiorophene. Food Cosmet. Toxicol. 11,625 (1973). 3. Greenwood, N.D., Hetherington, G., Curliffe, W.J., et al., An evaluation of the gas chromatographic estimation of trace quantities of hexachlorophene. J. Chromatogr. 89, 103 (1974). 4. Calesnick, B., Costello, C.H., Ryan, J.P., and DiGregorio, G.J., Percutaneous absorption of hexachlorophene following daily whole body washings. Toxicol. Appi. Pharmacol. 32, 204 (1975). 5. Browning, R.S., Greco, J., and Warrington, H.P., Gas chromatographic determination of hexachlorophene in blood and urine. J. Pharm. Sci. 57, 2165 (1968)6. Ferry, D.G., and McQueen, E.G., Hexachlorophene analysis in blood by electron capture gas chromatography. J. Chromatogr. 76, 233 (1973). 7. Plueckhahn, V.D., Infant antiseptic skin care and hexachlorophene. Med. J. Aust. 1,93 (1973). 8. Porcaro, P.J., and Shubiak, P., Detection of subnanogram quantities of hexachiorophene by electron capture gas chromatography. Anal. Chem. 40, 1232 (1968). . McLafferty, F.W., Interpretation of Mass Spectra, W.A. Benjamin, Inc., London, 1973, p 19. 10. Curley, A., and Hawk, R., Hexachiorophene. I. Analysis in body fluids and tissues of experimental animals. Paper read at 161st National Meeting of the American Chemical Society, Los Angeles, March 28 to April 2, 1971. 11. Shuman, R.M., Leech, R.W., and Alvord, E.G., Neurotoxicity of hexachlorophene in the human. 1. A clinicopathologic study of 248 children. Pediatrics 54,689 (1974).

CLINICAL CHEMISTRY,

Vol. 23, No. 6, 1977

947