Simultaneous isothermal determination of diphenylhydantoin and phenobarbital serum levels by gas-liquid chromatography following flash-heater hexylation

Clinica Chimica Acta, 67 (1976) 7-13 0 Elsevier Scientific Publishing Company,

Amsterdam

-Printed

in The Netherlands

CCA 7420

SIMULTANEOUS ISOTHERMAL DETERMINATION OF DIPHENYLHYDANTOIN AND PHENOBARBITAL BY GAS-LIQUID CHROMATOGRAPHY FOLLOWING HEXYLATION

THOMAS

J. GIOVANNIELLO

and JOSEPH

SERUM LEVELS FLASH-HEATER

PECCI *

Laboratory Service and the Surgical Research Unit, Veterans Admjnistratio~ Boston, Mass. 02130 (U.S.A.) (Received

Hospital,

July 7, 1975)

Summary A rapid, accurate, precise, and sensitive procedure for the simultaneous determination of diphenylhydantoin and phenobarbital under isothermal conditions involves on-column hexylation of the compounds. After extraction of serum samples, the evaporated residues are dissolved in tetr~exylammonium hydroxide. An aliquot of the resulting solution is introduced directly into a gas chromatograph where conversion to hexyl derivatives and subsequent separation takes place. The hexylated derivatives are identified by their retention times relative to appropriate internal standards. Concentrations are obtained from standard curves plotting relative peak height versus concentration of drug.

Introduction Diphenylhydantoin (DPH) is one of the most effective and most widely used of the anticonvulsant agents currently in use. This drug is metabolized quite differently from one patient to another. Because of this, periodic monitoring of blood levels is desirable in order to regulate drug dosage for optimum control of seizures. There have been numerous methods reported for determining blood levels of DPH [l] . It is generally agreed that of all the techniques presently available for determining DPH levels, those utilizing gas chromato~aphy [ 2-101 are superior. We have been determining DPH serum levels according to the method re* Address reprint requests to: Boston. Mass. 02130, U.S.A.

Joseph Pecci. VA

Hospital (151A).

150

South Huntinkton Ave..

ported by Barrett 161. In this method, analyses are carried out by methylating DPH at the hot injection point of a gas chromatographic column utilizing 5-(pmethylphenyl)-5-phenylhydantoin (MPPH) as the internal standard. The column temperature of 250°C causes phenobarbital (PB) to be eluted too early in the run to be of any analytical value, Since PB is a drug often used concomitantfy with DPH in the treatment of epileptic patients, we have been required to determine its presence in serum samples along with DPH. Up until recently, we have been carrying out barbiturate analysis separately using a spectrophotometric technique for total barbiturate [ 111. Several methods for the simultaneous gas chromatographic determination of DPH and PB have been reported [12-221 but none was adopted by us since they had one or more of the following shortcomings: (a) column packings other than the versatile 3% OV-17 on GasChrom Q were necessary; (b) temperature pro~amn?ing was required; (cf analyses were carried out at low temperatures necessitating ‘tong periods of time for analyses; fdf derivatization techniques were not used; (e) mare than one peak was seen if PB was derivatized; (f) an internal standard was not used. We felt that the capability for determining PB concentrations along with our DPW analyses would be highly desirable. To do this, it is only necessary to increase the retention time of PB while incorporating a suitable internal standard for PB. From our recent study f23] I it became clear that increasing the retention time of PB could be accomplished by merely lengthening the aikyl chain in its derivative. Specifically, hexylation of both PB and DPH gave rise to compaunds whose retention times were favorable for analytical use. We therefore devised a technique allowing us to extend the DPH analysis to include PB by hexylating rather than methylating each compound. We selected the recently reported 5-ethyl-5.~-t~iylbarbituric acid (ETBA) as an internal standard for PB f24,25]. No other changes in the DPH analysis were required. Materials and methods

Reagents Tetrahexylammonium hydroxide (THexAH), 0.2 molll in methanol *. Dissolve 0.02 mol of tetrahexylammonium iodide (Eastman Kodak Co., Rochester, N.Y.) in 100 mf of absolute methanol. Add 3.5 g of finely divided silver oxide to the methanolie solution and mix the resulting slurry for at least one hour at 20°C to precipitate silver iodide completely. Filter the mixture and store the filtrate consisting of 0.2 mol/l THexAH in well-stoppered, amber-colored glass bottles. We found this solution to be stable for at least 3 months when stored at room temperature. DPHfPB standard stoch ~o~~t~o~. DPH, over 99% pure, was obtained from the Aldrich Chemical Co., Milwaukee, Wise. and PB, U.S.P. was obtained from Ma~inckrodt Chemical Works, St. Louis, MO. 100 mg each of DPH and PB were * Available ~omrnerciaUy from Milton Laboratory U.S.A.

Products, 75 Adams St., Milton. Mass. 02186,

9

dissolved in 100 ml of absolute ethanol. This stock solution was stored at 4°C for periods no longer than three months. DPHIPB standard working solutions. 0.5 to 10.0 ml aliquots of the stock DPH/PB standard solution were each diluted to 100 ml with distilled water to provide solutions containing from 5 to 100 pg of DPH and PB per ml. These working solutions were prepared fresh just prior to use. ETBAIMPPH internal standard stock solution. ETBA and MPPH, both over 99% pure, were obtained from the Aldrich Chemical Co., Milwaukee, Wise. 450 mg of ETBA and 150 mg of MPPH were dissolved in 200 ml of chloroform. This stock solution was stored at 4°C for no longer than three months. ETBAIMPPH internal standard working solution. 0.5 ml of the stock ETBA/ MPPH standard solution was dissolved in 250 ml of chloroform to provide a solution containing 4.5 l.(g of ETBA and 1.5 pg of MPPH per ml. Apparatus Gas chromatography was carried out using a Perkin-Elmer Model 3920 instrument equipped with a hydrogen flame ionization detector and a PerkinElmer Model 56 recorder with a range of O-l mV and a chart speed of 5 mm per min. Glass columns 6-ft long by l/4-inch o.d. packed with 3% OV-17 on GasChrom Q, 100-120 mesh, were used. The columns were conditioned by heating them at 100°C for 1 h and increasing the temperature at the rate of l”C/min until the temperature reached 275”C, at which it was maintained for 18 h. The detector end of the column was disconnected during the conditioning period. The operating conditions were as follows: nitrogen carrier gas at a flow rate of 30 ml/min; hydrogen produced by a Protran 150-A generator (Trienco, Raleigh, N.C.) at 21 psi; air at 50 psi; injector temperature, 300°C; oven temperature, 250°C; detector temperature, 300°C; attenuation, X 16; range, X 10; recorder, 1 mV full-scale. Procedure

In 40-ml glass centrifuge tubes with screw caps having Teflon-faced rubber liners, add 1.0 ml each of DPH/PB standard working solutions and the serum samples. Then add 1 ml of 1 N hydrochloric acid and 7.0 ml of the ETBA/ MPPH internal standard working solution. Shake the mixture well for one minute and centrifuge to separate the phases. If a wafer of insoluble material is present at the interface, it can be lifted out carefully with a spatula. Transfer 5 ml of 0.45 N sodium hydroxide to the chloroform phase in the tube and shake for 1 min. Centrifuge and aspirate the bottom chloroform layer. Add 1 ml of 6 N hydrochloric acid to the aqueous phase now remaining in the tube, mix thoroughly and then add 10 ml of chloroform. Shake for 1 min, centrifuge, and aspirate the top aqueous layer. Transfer about -7 ml of the remaining chloroform layer into a 12-ml glass conical centrifuge tube. Evaporate the chloroform to dryness by directing a stream of nitrogen over the chloroform while the tubes are immersed in a water bath at 35 to 40°C. When the samples have been evaporated almost to dryness, wash down the walls of the centrifuge tube with 0.5 to 1 ml of chloroform and evaporate the sample to dryness. Dis-

solve the residue with 25 ~1 of methanolic 0.2 mol/l THexAH 1 to 2 1.11of the resulting solution into the gas chromatograph.

and inject from

Results Fig. 1 presents a gas chromatogram showing the results obtained when a mixture of PB, ETBA, DPH, and MPPH are extracted and hexylated according to the procedure described. The resulting peaks are symmetrical with PB emerging from the column first and well removed from the solvent front, followed by ETBA, DPH, and MPPH in that order. Table I summarizes the retention and relative retention times of the compounds all of which are eluted from the column within 15 min after injection under the conditions outlined. Standard solutions containing varying amounts of PB and DPH were analyzed. The ratio, R, was calculated by dividing the hexylated PB and DPH peak heights by the peak height of their corresponding derivatized internal standards. These R values were plotted as a function of concentration. Sample losses during extraction and other manipulations are compensated for by the presence of the internal standards since the values of R are not affected. As can be seen in Fig. 2, the plot is linear up to a concentration of 100 pg/ml for PB and up to 70 pg/ml for DPH. For bobh drugs, the lower limit of detection is 1 pg/ml. When unknown serum samples were analyzed relative retention times

Fig.

1.

Typical

gas chromatogram

of

hexylated

derivatives

of

PB (1).

ETBA

(2).

DPH

(3).

and

MPPH

(4).

11

TABLE 1 SUMMARY

OF RETENTION

RETENTION

PB ETBA

TIMES

Relative retention time

Retention time (min)

Compound

DPH MPPH __ ____--

AND RELATIVE

5.8 1.2

0.81 -

10.6 13.6

0.78 -

__-

TABLE II DAY-TO-DAY POOLS Day

AND WITHIN-DAY

VARIATION

OF PB AND DPH ANALYSES

USING TWO SERUM

DPH (/x/d)

PB M/ml) a.m.

p.m.

a.m.

p.m.

a.m.

p.m.

a.m.

p.m.

7.8 1.8 1.6 7.8 7.5 1.7 1.6 7.6 1.8 7.4 7.3 1.2 1.2 7.7 1.6 7.6

7.9 1.6 7.5 7.8 7.3 7.6 7.7 7.5 7.7 7.4 1.2 1.0 7.4 7.6 7.5 7.7

14.9 14.2 14.0 14.6 14.4 14.4 14.8 14.8 14.9 14.2 14.4 14.4 14.2 14.6 14.8 14.5

14.9 14.9 14.4 14.4 14.6 14.5 14.8 14.8 14.8 14.0 14.6 14.5 14.5 14.5 14.7 14.5

7.6 7.7 7.8 8.1 8.0 7.5 7.6 6.9 7.2 7.4 7.4 7.4 7.4 7.5 7.3 7.2

7.2 7.7 8.1 8.2 8.1 7.4 7.4 7.5 7.4 7.5 7.3 7.1 1.2 1.2 7.4 7.4

14.0 14.8 14.0 15.0 14.9 14.2 14.0 15.2 14.8 14.6 14.5 14.2 14.5 14.5 14.2 14.2

14.2 14.8 14.5 14.2 15.2 14.1 14.2 14.2 14.0 14.6 14.5 14.4 14.8 14.5 14.5 14.2

Mean

7.6

7.5

14.5

14.6

1.5

7.5

14.5

14.5

S.D.

0.2

0.2

0.3

0.2

0.3

0.3

0.4

0.3

C.V. (a)

2.1

3.0

1.9

1.6

4.0

4.6

2.6

2.1

1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16

TABLE III ACCURACY

OF PB AND DPH DETERMINATIONS

Amount PB and DPH added (f&ml)

5.0 8.0 10.0 15.0 20.0 40.0 50.0

~-_--

Amount determined (&z/ml)

Percentage of amount expected -

PB

DPH

PB

DPH

5.1 8.2 10.3 14.9 19.5 39.4 49.9

5.1 8.0 10.0 15.2 20.0 40.0 48.2

102 103 103 99.3 97.5 98.5 99.8

102 100 100 101 100 100 96.4

12

-/3

-f2

-/I

-/o

-9

-8

Qz

-7

-6

-5

-4

-3

0

D

20

30

40

CONCEN Fig.

2.

Standard

curves

50 7RATIoN

in which

60 70 f,ughf)

80

K is plotted

90

fO0

as a function

of concentration

for PB (

w

) and

DPH

(0-j.

were calculated to identify the peaks as corresponding to hexylated PB and DPH. Values for R were then calculated and concentrations were read directly from standard curves. Standard curve determinations were made on a daily basis since minor day-to-day variations were noted. Two serum pools were aliquoted into portions, each of which were stored frozen. Analyses were performed on these aliquots on a day-to-day as well as a within-day basis. The results are summarized in Table II. Known amounts of PB

TABLE

IV

COMPARISON

OF

RESULTS

OBTAINED

HEXYLATION DPH

PB (/s/d) -__ Hexylation

BY

-~ Methyl&ion

METHYLATION

TECHNIQUES _-__

(/.WmI)

-

% Deviation ,O

AND

Hexylation

Methylation

% Deviation

7.5

7.5

2.2

2.2

0

10.2

11.0

7.8

4.8

5.1

6.3

14.5

14.8

2.1

7.8

7.7

1.3

17.2

16.0

7.5

8.8

8.8

0

29.4

29.0

1.4

14.5

14.8

2.1

31.2

29.0

7.6

23.1

22.9

0.9

39.0

38.4

1.6

29.1

28.8

1.0

42.9

43.1

0.4

40.0

38.5

3.9

13

and DPH were added to drug-free human serum. Analyses were carried out for each over a broad concentration range. The results are tabulated in Table III. Results of analyses obtained for PB and DPH carried out simultaneously using the hexylation method outlined in this paper were compared to results obtained from methods utilizing separate methylation of PB [26] and DPH [6]. The results of this study are presented in Table IV. Discussion The low standard deviations and coefficients of variation in Table II indicate that the reproducibility of the hexylation method is very good. The data presented in Table III show the method to be quite accurate since the values determined for PB and DPH are very close to the values for the actual amount added to serum. The agreement in values obtained when analyzing the same samples by another method (Table IV) is excellent. We have shown that it is possible to simultaneously determine PB and DPH under isotherms conditions by on-column hexylation. Both compounds are eluted from the gas chromato~aphic column in less than 15 min. Hexylation of PB eliminates the formation of questionable “early-phenobarbital” as reported in other published methods [ 19,22,27,28]. The particular advantage of isothermal determination is that it eliminates the need of time-consuming temperature programming. Also, those laboratories having gas chromatographs in which the temperature programming feature is lacking, may now be able to perform simultaneous analyses of PB and DPH. References 1 Pertry, J.K., Smith, L.D. and White, M.S.P.H. (1972) DHEW Publication No. (NIH) 73-396. intendant of Documents, U.S. Government Printing Office, Washington, D.C. 20402 2 Sandberg, D.H., Resnick, G.L. and BacaIIao, C.Z. (1968) Anal. Chem, 40, 736 3 Sabih, K. and Sabih, K. (1969) Anal. Chem. 41.1452 4 Chang, T. and Glazko, A.J. (1970) J. Lab. Clin. Med. 75,145 5 MacGee, J. (1970) Anal. Chem. 42. 421 6 Barrett, M.J. (1971) Ciin. Chem. Newsletter 3.16 7 Hammer. R.H.. WiIder, B.J., Streiff, R.R. and Mayersdorf, A. (1971) J. Pbarm. Sci. 60. 327 8 Larsen, N.E. (1971) Med. Lab. Tech. 28,377 9 Chin, D., Fastlick, E. and Davidow. B. (1972) J. Chromatogr. 71, 545 10 Estas, A. and Dumont, P.A. (1973) J. Chromatogr. 82. 307 11 WilIiams,,L.A. and Zak. B. (1959) Clin. Chem. Acta 4.170 12 Pippenger. C.E. and GiBen, H.W. (1969) Clin. Chem. 15. 582 13 Baylis, E.M., Fry, D.E. and Marks, V. (1970) Clin. Chim. Acta 30.93 14 Kupferberg, H.J. (1970) Clin. Chem. Acta 29. 283 15 Van Meter, J.C., Buckmaster, H.S. and Shelley, L,L. (1970) Clin. Chem. 16,135 16 Meiier, J.W.A. (1971) Epilepsia 12, 341 17 Cooper, R.G., Greaves, M.S. and Owen. G. (19’72) CIIn. Chem. 18,1343 18 Cremers, H.M.H.G. and Verheesen. P.E. (1973) Clin. Chim. Acta 48,113 19 PerchaIski, R.J., Scott, K.N., Wilder, B.J. and Hammer, R.H. (1973) J. Pharm. Sci. 62,1735 20 Roger, J.C.. Rodgers, G. and Soo, A. (1973) Clin. Chem. 19. 590 21 Goudie, J.H. and Burnett, D. (1974) Clin. Chim. Acta 43,423 22 Osiewicz, R.. Aggarwal, V., Young, R.M. and Sunshine, I. (1974) J. Chromatogr. 88.157 23 Pecci, J. and GiovannieIIo, T.J. (1975) J. Chromatogr. 109.163 24 Gibbs, E.L. and Gibbs, T.J. (1974) Gibbs Laboratory Reports. Part A, Method PHA-TOL-73, ary 20 25 Solow, E.B.. Metaxas, J.M. and Summers, T.R. (1974) J. Chromatogr. Sci. 12,256 26 Barrett. M.J. (1971) Clin. Chem. Newsletter 3,1 27 Kananen, G., Osiewiez, R. and Sunshine. I. (1972) 3. Chromatogr. Sci. 10,283 28 Van Meter, J.C. and Gillen. H.W. (1973) Clin. Chem. 19,359

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