Vol. 11, No. 5
ANTYImCRODIAL AGENTS AND CnHMonHRAnY, May 1977, p. 914-915 Copyright C 1977 American Society for Microbiology
Printed in U.S.A.
Gas Chromatographic Determination of 5-Fluorocytosine: Modified Extraction Method
a
SIOK-HUI WEE AND JOHN P. ANHALT*
Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901
Received for publication 3 January 1977
A modified procedure for the gas-chromatographic determination of 5-fluorocytosine in serum is described. A more volatile solvent mixture for extraction is used to reduce sample preparation time before chromatography. Comparison of values from assay of 12 serum samples by our method and the reference method gave a correlation coefficient of 0.99.
Harding et al. reported the serum assay of
5-fluorocytosine by a gas-chromatographic method (1), which we shall refer to as the reference method. A lengthy step in that procedure is the evaporation of 1-butanol that is used for extraction of the drug. We have developed a modified procedure that uses a more volatile solvent mixture for extraction. A gas chromatograph (model 2740, Varian Aerograph, Walnut Creek, Calif.) equipped with a flame ionization detector was used. A glass column (183 cm by 2 mm, internal diameter) packed with 3% OV-17 on 100/120 Gas Chrom Q (Applied Sciences Labs, State College, Pa.) was used for analysis. The operating conditions were as follows: injection port temperature, 185°C; program oven temperature, 100°C (2 min) to 270°C at 200C/min; detector temperature, 285°C; carrier gas (nitrogen) flow rate, 22 mllmin; hydrogen flow rate, 25 ml/min; and air flow rate, 330 ml/min. The suppliers for the following reagents were: 5-fluorocytosine, Hoffmann LaRoche, Nutley, N.J.; cytosine, Aldrich Chemical Co., Milwaukee, Wis.; and Regisil, Regis Chemical Co., Morton Grove, Ill. Method I. A mixture of 200 ,ul of serum and 1.8 ml of 0.4 M sodium phosphate buffer, pH 7.4, containing 10 ,g of internal standard, cytosine, was washed with 5 ml of methylene chloride by shaking for 5 min in a Labquake shaker (Labindustries, Berkeley, Calif.). Phases were separated by centrifuging for 10 min at 1,000 x g. The aqueous upper layer was then extracted with 5 ml of 2-propanol-methylene chloride (3:1, vol/vol), shaken and centrifuged as above. The organic upper layer was transferred to a conical test tube and evaporated to dryness under a stream of air at 550C for between 2 and 3 h.
Method II. Serum, 200 4ul, was mixed with 2 ml of 0.1 M sodium phosphate buffer, pH 2, containing 10 gig of cytosine. The mixture was then extracted with 5 ml of 2-propanol-methylene chloride (2:1, vol/vol) for 5 min and centrifuged as in method I. The aqueous top layer was neutralized by the addition of 1 ml of 1 M sodium phosphate buffer, pH 7.9. The mixture was extracted with 5 ml of 2-propanol-methylene chloride (2:1, vol/vol), and the organic top layer was removed and evaporated to dryness. The residue in each case was silylated with 20 ~ .10
0
1.5 F
;P..
U)
1.0p.
0
'I3
4.5p. 40
20
60
80
5 Fluorocytosine, pg /mi -
FIG. 1. Standard curve for 5-fluorocytosine in serum by method 1.
50 gul of acetonitrile-Regisil (2:1, vol/vol) mixture, mixed for 5 min in an ultrasonic vibrator, and allowed to stand at room temperature for 25 min before injection. Serum standards were prepared as per Harding et al. (1). Typical standard curves are shown in Fig. 1 and 2. In the recovery study of 5-fluorocytosine from serum, the internal standard was omitted from the buffer but was added to the organic extract 914
NOTES
VOL. 11, 1977
before e vaporation. The peak height of 5-fluorocytosine was then measured against that of cytosinet. Recovery was 74% from method I and 45% fro]m method II. 2.0 1.5
~*
1.0
0.5
Z/00".20
40
60
80
5 Fluorocytosine, pg /mli FIG. 2 Standard curve for 5-fl uorocytosine in serum by ,nethod II.
_-
100
,%
80
0
4E60
Q)
S.
.S 0
./.
v
4
;:
20
915
Twelve serum samples, which were provided by H. Solomon (Johns Hopkins Hospital, Baltimore, Md.), were analyzed in duplicate by method I and by Solomon, using the reference method. The mean values obtained were regressed against their results. The slope of the linear least squares line was 0.91, the intercept was -1.94, and the correlation coefficient was 0.99 (Fig. 3). Precision was estimated from the differences observed between the duplicate determinations. The mean difference was 4.5%. The maximum difference observed was 10%, which was obtained from a specimen that contained 1.1 ,ug of 5-fluorocytosine per ml. Method II was developed after two serum samples were found to contain a peak that could possibly interfere with the accurate measurement of cytosine. This peak was eliminated by acid washing before extraction of 5-fluorocytosine. However, in these two samples, our results with method I still corresponded well with those obtained by the reference method. Duplicate determinations on serum standards by method II showed a mean difference between duplicates of 2.7% (n = 4). The reference method used butanol as an extraction solvent. We found that 2-propanolmethylene chloride effectively reduced the evaporation time from between 4 and 5 h to between 2 and 3 h. Also, method II conveniently resulted with the desired phase in the upper layer, that is, aqueous in the first extraction and organic in the second extraction. This change in solvent also gave a better recovery. We thank Harvey M. Solomon, Johns Hopkins Hospital, Baltimore, Md., for providing the serum samples for our assay and the data from their assay for the evaluation of our methods.
20 5-
40
60
80
100
Fluorocytosine (reference method), pg/mi
FIG. 3. Comparison of gas-chromatographic assay of 5-fluorocytosine in serum by reference method and method I.
LITERATURE CITED 1. Harding, S. A., G. F. Johnson, and H. M. Solomon. 1976. Gas-chromatographic determination of 5-fluorocytosine in human serum. Clin. Chem. 22:772-776.
ANTYImCRODIAL AGENTS AND CnHMonHRAnY, May 1977, p. 914-915 Copyright C 1977 American Society for Microbiology
Printed in U.S.A.
Gas Chromatographic Determination of 5-Fluorocytosine: Modified Extraction Method
a
SIOK-HUI WEE AND JOHN P. ANHALT*
Section of Clinical Microbiology, Department of Laboratory Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901
Received for publication 3 January 1977
A modified procedure for the gas-chromatographic determination of 5-fluorocytosine in serum is described. A more volatile solvent mixture for extraction is used to reduce sample preparation time before chromatography. Comparison of values from assay of 12 serum samples by our method and the reference method gave a correlation coefficient of 0.99.
Harding et al. reported the serum assay of
5-fluorocytosine by a gas-chromatographic method (1), which we shall refer to as the reference method. A lengthy step in that procedure is the evaporation of 1-butanol that is used for extraction of the drug. We have developed a modified procedure that uses a more volatile solvent mixture for extraction. A gas chromatograph (model 2740, Varian Aerograph, Walnut Creek, Calif.) equipped with a flame ionization detector was used. A glass column (183 cm by 2 mm, internal diameter) packed with 3% OV-17 on 100/120 Gas Chrom Q (Applied Sciences Labs, State College, Pa.) was used for analysis. The operating conditions were as follows: injection port temperature, 185°C; program oven temperature, 100°C (2 min) to 270°C at 200C/min; detector temperature, 285°C; carrier gas (nitrogen) flow rate, 22 mllmin; hydrogen flow rate, 25 ml/min; and air flow rate, 330 ml/min. The suppliers for the following reagents were: 5-fluorocytosine, Hoffmann LaRoche, Nutley, N.J.; cytosine, Aldrich Chemical Co., Milwaukee, Wis.; and Regisil, Regis Chemical Co., Morton Grove, Ill. Method I. A mixture of 200 ,ul of serum and 1.8 ml of 0.4 M sodium phosphate buffer, pH 7.4, containing 10 ,g of internal standard, cytosine, was washed with 5 ml of methylene chloride by shaking for 5 min in a Labquake shaker (Labindustries, Berkeley, Calif.). Phases were separated by centrifuging for 10 min at 1,000 x g. The aqueous upper layer was then extracted with 5 ml of 2-propanol-methylene chloride (3:1, vol/vol), shaken and centrifuged as above. The organic upper layer was transferred to a conical test tube and evaporated to dryness under a stream of air at 550C for between 2 and 3 h.
Method II. Serum, 200 4ul, was mixed with 2 ml of 0.1 M sodium phosphate buffer, pH 2, containing 10 gig of cytosine. The mixture was then extracted with 5 ml of 2-propanol-methylene chloride (2:1, vol/vol) for 5 min and centrifuged as in method I. The aqueous top layer was neutralized by the addition of 1 ml of 1 M sodium phosphate buffer, pH 7.9. The mixture was extracted with 5 ml of 2-propanol-methylene chloride (2:1, vol/vol), and the organic top layer was removed and evaporated to dryness. The residue in each case was silylated with 20 ~ .10
0
1.5 F
;P..
U)
1.0p.
0
'I3
4.5p. 40
20
60
80
5 Fluorocytosine, pg /mi -
FIG. 1. Standard curve for 5-fluorocytosine in serum by method 1.
50 gul of acetonitrile-Regisil (2:1, vol/vol) mixture, mixed for 5 min in an ultrasonic vibrator, and allowed to stand at room temperature for 25 min before injection. Serum standards were prepared as per Harding et al. (1). Typical standard curves are shown in Fig. 1 and 2. In the recovery study of 5-fluorocytosine from serum, the internal standard was omitted from the buffer but was added to the organic extract 914
NOTES
VOL. 11, 1977
before e vaporation. The peak height of 5-fluorocytosine was then measured against that of cytosinet. Recovery was 74% from method I and 45% fro]m method II. 2.0 1.5
~*
1.0
0.5
Z/00".20
40
60
80
5 Fluorocytosine, pg /mli FIG. 2 Standard curve for 5-fl uorocytosine in serum by ,nethod II.
_-
100
,%
80
0
4E60
Q)
S.
.S 0
./.
v
4
;:
20
915
Twelve serum samples, which were provided by H. Solomon (Johns Hopkins Hospital, Baltimore, Md.), were analyzed in duplicate by method I and by Solomon, using the reference method. The mean values obtained were regressed against their results. The slope of the linear least squares line was 0.91, the intercept was -1.94, and the correlation coefficient was 0.99 (Fig. 3). Precision was estimated from the differences observed between the duplicate determinations. The mean difference was 4.5%. The maximum difference observed was 10%, which was obtained from a specimen that contained 1.1 ,ug of 5-fluorocytosine per ml. Method II was developed after two serum samples were found to contain a peak that could possibly interfere with the accurate measurement of cytosine. This peak was eliminated by acid washing before extraction of 5-fluorocytosine. However, in these two samples, our results with method I still corresponded well with those obtained by the reference method. Duplicate determinations on serum standards by method II showed a mean difference between duplicates of 2.7% (n = 4). The reference method used butanol as an extraction solvent. We found that 2-propanolmethylene chloride effectively reduced the evaporation time from between 4 and 5 h to between 2 and 3 h. Also, method II conveniently resulted with the desired phase in the upper layer, that is, aqueous in the first extraction and organic in the second extraction. This change in solvent also gave a better recovery. We thank Harvey M. Solomon, Johns Hopkins Hospital, Baltimore, Md., for providing the serum samples for our assay and the data from their assay for the evaluation of our methods.
20 5-
40
60
80
100
Fluorocytosine (reference method), pg/mi
FIG. 3. Comparison of gas-chromatographic assay of 5-fluorocytosine in serum by reference method and method I.
LITERATURE CITED 1. Harding, S. A., G. F. Johnson, and H. M. Solomon. 1976. Gas-chromatographic determination of 5-fluorocytosine in human serum. Clin. Chem. 22:772-776.
