Technical note
A SIMPLE RAPID GASCHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF THEOPHYLLINE IN DRIED WHOLE BLOOD ON FILTER PAPER CARDS M. Albani1 and P. A. Toseland2
In 1973 Kuzemko proposed theophylline for treatment of apnea in preterm infants (1). Ever since it has been widely used for this purpose and its therapeutic benefit for preventing apnea has been established (2, 3). However, pharmacokinetic studies revealed a biological half life several times longer in preterm infants (4) than in older children and adults (5). Therefore, routine monitoring of theophylline blood levels seems essential, the more so since little is known about potentially harmful effects on the developing organism (6). Using a nitrogen-specific detector a rapid simple gaschromatographic method is described which requires for the determination of theophylline only about 35 mg dried whole blood on filter paper cards obtained by capillary blood sampling technique.
Method and materials Filter paper cards as used in all nurseries for the Guthrie-test were stained with capillary blood. After drying at room temperature 3 ul of the Received: January 20, 1978
internal standard solution were added to each blood spot. Three to six spots punched out from the card were then extracted with 500 ul of a 0.45m phosphate buffer pH 4.5 and 5 ml of destilled ether in a glass stoppered by shaking for 10 min. After the sample was centrifuged the ether transfered to another tube was taken to dryness in a waterbath at 60° C under a stream of nitrogen. The residue was disolved in 15 ^1 °f methanol and 2 ul were injected into the gaschromatograph. Stock solutions Theophylline: 2.484 g EUPHYLLIN® (8O.5%> Theophylline and 19.5°/o Aethylendiamine) was disolved in 10 ml of water. 500 [A of this solution was disolved in 100 ml of methanol giving a concentration of 1 fx% theophylline/,ttl. With fresh heparinized drug-free blood containing 10 ,ag/ml the precision and variation studies were performed. In the same way the* linearity of the method was studied by adding 2.5 — 5.0 — 10.0 — 15 — 20 — 30 — 50,ug to 1 ml of drug free heparinized blood. Internal standard: Heptabarbital, a generous gift of CIBA-GEIGY, was disolved in methanol to a concentration of 100 ng/^tl.
Accepted: February 10, 1978
Address: M. A., Universitatskinderklinik, Humboldtallee 38, D-3400 GSttingen/Geimany Acknowledgement: This work was supported by a grant from the Deutsche Forschungsgemeinschaft SFB 33.
97
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1 University Children's Hospital Gottingen, F. R. G., and 2Dept. Clinical Chemistry, Guys' Hospital, London U. K.
GLC - conditions A dual-column Gaschromatograph Hewlett-Packard 5750 B with a nitrogen-specific flame -ionization-detector type 1516 A was used. Column: glass 4 ft 2 mm i.d. filled with O.50/0 HIEFF 8 BP on Chromosorb W HI' (Applied Science.) 80-1 00 mesh. Flowrates: air 180 mllmin, hydrogen 28 mllmin, carrier gas (helium) 40 mllmin. Temperatures: oven 240" C, injection port 300" C, detector 370" C. The column was conditioned in the following manner: After 30 min at room temperature under carrier gas to remove all oxygen from the column it was heated up with 2"Imin to 275" C and stayed there for 20 min. After cooling down the conditioning procedure was repeated.
Vol. 9, No. 1, 1978
1 Neuropidiiltrie
whole range with a correlation coefficient of r = 0.9784. The analysis of ten samples with identical concentration at ten successive days revealed a day-to-day variance of c! 2.9O/o and no significant influence of storing the blood-stained cards at room temperature up to ten days. Since blood viscosity and therefore the amount of blood required to stain the whole preprinted spot on the card essentially depends upon the hematocrit we studied the influence of hematocrit values between 10-900/0. The results indicated a marked effect on the determination at hematocrit values below 30°/o and above 60°/n. In the "normal" range between 3060°/o the variation caused by different hematocrit values did not exceed 100/0 so that for clinical purposes a corrective calculation can be neglected.
Results The precision of the method studied by simultaneous analysis of ten different samples with identical concentration revealed a coefficient of variation of 5.2OIo (range 6.7-3.8O/o in five subsequent investigations, two with prepared drug-free blood, three with pooled blood of theophylline treated patients). There was a steep increase in variation of the results, when the spots preprinted on the cards were not completely stained with blood. Thus, attention has to be paid to a careful blood sampling as the most important factor for a precise and reliable determination. The method is linear over the
Discussion Methods thus far described for the measurement of theophyiline blood levels carry the disadvantage of large sample size (7), methylation (8) or time consuming and complicated extraction and/or analysis procedures (9, 10). Small sample volume is inevitable in preterm infants and methylation of theophylline is inappropriate since thereby caffeine is produced which may be already present in blood of breast-fed infants. Therefore, the determination without forming a derivative has obvious methodological advantages. Using the high specifity and
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Albani and Toseland
Micromethod for theophylline determination
Similar problems occur during theophylline treatment of children (5) of older age groups. In an editorial Bierman (13) recently stated that the development of micromethods for the determination of theophylline would help to solve the problems of tailoring the dose to thepatients' individual drug requirement. As we were able to show that the method described is unaffected by storing the samples at room temperature for several days as well as by hematocrit differences in the "normal" range this determination procedure allows an accurate drug level monitoring in all pediatric patients, even in those not being attached to a hospital with drug measurement facilities. Thus, this method offers the possibility of controlling theophylline blood concentrations combining the benefit of a rather atrahmic sampling of small amounts of blood with the advantage of sending the sample to a well equipped and experienced laboratory.
Literature 1. Kuzemko, J. A., Paala, I.: Apnoeic attacks 2.
3.
4.
5.
in the newborn treated with aminophylline. Arch. Dis. Child. 48: 404 (1973). Shannon, D. C., Gotay, F., Stein, I. M. et al.: Prevention of apnea and bradycardia in low-birthweight infants. Pediatrics 55: 589 (1975). Uauy, R., Shapiro, D. L., Smith, B.: Treatment of severe apnea in prematures with orally administered theophylline. Pediatrics 55: 595 (1975). Aranda, J. V., Sitar, D. S., Parsons, W. D. et al.: Pharmacokinetic aspects of theophylline in premature newborns. New Engl. J. Med. 295: 413 (1976). Ellis, E. E., Koysooko, R., Levy, G . : Pharmacokinetics of theophylline in children with asthma. Pediatrics 58: 542
(1976). 6. Lucey, J. F.: The xanthine treatment of apnea of prematurity. Pediatrics 55: 584 (1975). 7. Wesley-Hadzija, B., Mattocks, A. M.:
Specific thin-layer chromatographic method for the determination of theophylline in plasma in the presence of other drugs. J. Chromatogr. 115: 501 (1975). 8. Brockmann-Hanssen, E.,Olawuyi-Oke, T.: Gaschromatography of barbiturates, phenolic alkaloids and xanthine bases: Flashheater methypation by means of trimethylanilinium hydroxide. J. Pharm. Sci. 58: 370 (1969). 9. Thompson, R. D., Nagasawa, H. T., Jenne, J. W.: Determination of theophylline and
its metabolites in human urine and serum by high-pressure-liquid chromatography. J. Lab. Clin. Med. 84: 584 (1974). 10. Cook, G. L., Twine, M. E., Myers, M. et al.: Theophylline radioimmunassay. Res. Com. Chem. Pathol. Pharmacol. 13: 497 (1976). 11. Toxland, P. A., Oldigs, H. D.: The
determination of anticonvulsant drugs in dried whole blood samples on filter paper cards. Epileptology 1976 ed. D. Janz, publ. Thieme - Stuttgart, p. 416. 12. Albani, M., Toseland, P. A., Oldigs, H. D.: A new methodological approach to routine monitoring of anticonvulsant drugs. In preparation. 13. Bierman, C. W.: Theophylline in asthma. Pediatrics 58: 623 (1976).
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selectivity of a nitrogen detector theophylline can now be readily chromatographed as the free compound in a crude ether extract of 35 mg whole blood on filter paper cards giving a result within about 20 minutes. The way of blood sampling has already been described for phenobarbitone (1 1) and has been made applicable in the meantime for the determination of several other anticonvulsant drugs (12). Now being used for more than three months the method has been proven advantageous for controlling theophylline drug levels in preterm infants.
A SIMPLE RAPID GASCHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF THEOPHYLLINE IN DRIED WHOLE BLOOD ON FILTER PAPER CARDS M. Albani1 and P. A. Toseland2
In 1973 Kuzemko proposed theophylline for treatment of apnea in preterm infants (1). Ever since it has been widely used for this purpose and its therapeutic benefit for preventing apnea has been established (2, 3). However, pharmacokinetic studies revealed a biological half life several times longer in preterm infants (4) than in older children and adults (5). Therefore, routine monitoring of theophylline blood levels seems essential, the more so since little is known about potentially harmful effects on the developing organism (6). Using a nitrogen-specific detector a rapid simple gaschromatographic method is described which requires for the determination of theophylline only about 35 mg dried whole blood on filter paper cards obtained by capillary blood sampling technique.
Method and materials Filter paper cards as used in all nurseries for the Guthrie-test were stained with capillary blood. After drying at room temperature 3 ul of the Received: January 20, 1978
internal standard solution were added to each blood spot. Three to six spots punched out from the card were then extracted with 500 ul of a 0.45m phosphate buffer pH 4.5 and 5 ml of destilled ether in a glass stoppered by shaking for 10 min. After the sample was centrifuged the ether transfered to another tube was taken to dryness in a waterbath at 60° C under a stream of nitrogen. The residue was disolved in 15 ^1 °f methanol and 2 ul were injected into the gaschromatograph. Stock solutions Theophylline: 2.484 g EUPHYLLIN® (8O.5%> Theophylline and 19.5°/o Aethylendiamine) was disolved in 10 ml of water. 500 [A of this solution was disolved in 100 ml of methanol giving a concentration of 1 fx% theophylline/,ttl. With fresh heparinized drug-free blood containing 10 ,ag/ml the precision and variation studies were performed. In the same way the* linearity of the method was studied by adding 2.5 — 5.0 — 10.0 — 15 — 20 — 30 — 50,ug to 1 ml of drug free heparinized blood. Internal standard: Heptabarbital, a generous gift of CIBA-GEIGY, was disolved in methanol to a concentration of 100 ng/^tl.
Accepted: February 10, 1978
Address: M. A., Universitatskinderklinik, Humboldtallee 38, D-3400 GSttingen/Geimany Acknowledgement: This work was supported by a grant from the Deutsche Forschungsgemeinschaft SFB 33.
97
Downloaded by: University of British Columbia. Copyrighted material.
1 University Children's Hospital Gottingen, F. R. G., and 2Dept. Clinical Chemistry, Guys' Hospital, London U. K.
GLC - conditions A dual-column Gaschromatograph Hewlett-Packard 5750 B with a nitrogen-specific flame -ionization-detector type 1516 A was used. Column: glass 4 ft 2 mm i.d. filled with O.50/0 HIEFF 8 BP on Chromosorb W HI' (Applied Science.) 80-1 00 mesh. Flowrates: air 180 mllmin, hydrogen 28 mllmin, carrier gas (helium) 40 mllmin. Temperatures: oven 240" C, injection port 300" C, detector 370" C. The column was conditioned in the following manner: After 30 min at room temperature under carrier gas to remove all oxygen from the column it was heated up with 2"Imin to 275" C and stayed there for 20 min. After cooling down the conditioning procedure was repeated.
Vol. 9, No. 1, 1978
1 Neuropidiiltrie
whole range with a correlation coefficient of r = 0.9784. The analysis of ten samples with identical concentration at ten successive days revealed a day-to-day variance of c! 2.9O/o and no significant influence of storing the blood-stained cards at room temperature up to ten days. Since blood viscosity and therefore the amount of blood required to stain the whole preprinted spot on the card essentially depends upon the hematocrit we studied the influence of hematocrit values between 10-900/0. The results indicated a marked effect on the determination at hematocrit values below 30°/o and above 60°/n. In the "normal" range between 3060°/o the variation caused by different hematocrit values did not exceed 100/0 so that for clinical purposes a corrective calculation can be neglected.
Results The precision of the method studied by simultaneous analysis of ten different samples with identical concentration revealed a coefficient of variation of 5.2OIo (range 6.7-3.8O/o in five subsequent investigations, two with prepared drug-free blood, three with pooled blood of theophylline treated patients). There was a steep increase in variation of the results, when the spots preprinted on the cards were not completely stained with blood. Thus, attention has to be paid to a careful blood sampling as the most important factor for a precise and reliable determination. The method is linear over the
Discussion Methods thus far described for the measurement of theophyiline blood levels carry the disadvantage of large sample size (7), methylation (8) or time consuming and complicated extraction and/or analysis procedures (9, 10). Small sample volume is inevitable in preterm infants and methylation of theophylline is inappropriate since thereby caffeine is produced which may be already present in blood of breast-fed infants. Therefore, the determination without forming a derivative has obvious methodological advantages. Using the high specifity and
Downloaded by: University of British Columbia. Copyrighted material.
Albani and Toseland
Micromethod for theophylline determination
Similar problems occur during theophylline treatment of children (5) of older age groups. In an editorial Bierman (13) recently stated that the development of micromethods for the determination of theophylline would help to solve the problems of tailoring the dose to thepatients' individual drug requirement. As we were able to show that the method described is unaffected by storing the samples at room temperature for several days as well as by hematocrit differences in the "normal" range this determination procedure allows an accurate drug level monitoring in all pediatric patients, even in those not being attached to a hospital with drug measurement facilities. Thus, this method offers the possibility of controlling theophylline blood concentrations combining the benefit of a rather atrahmic sampling of small amounts of blood with the advantage of sending the sample to a well equipped and experienced laboratory.
Literature 1. Kuzemko, J. A., Paala, I.: Apnoeic attacks 2.
3.
4.
5.
in the newborn treated with aminophylline. Arch. Dis. Child. 48: 404 (1973). Shannon, D. C., Gotay, F., Stein, I. M. et al.: Prevention of apnea and bradycardia in low-birthweight infants. Pediatrics 55: 589 (1975). Uauy, R., Shapiro, D. L., Smith, B.: Treatment of severe apnea in prematures with orally administered theophylline. Pediatrics 55: 595 (1975). Aranda, J. V., Sitar, D. S., Parsons, W. D. et al.: Pharmacokinetic aspects of theophylline in premature newborns. New Engl. J. Med. 295: 413 (1976). Ellis, E. E., Koysooko, R., Levy, G . : Pharmacokinetics of theophylline in children with asthma. Pediatrics 58: 542
(1976). 6. Lucey, J. F.: The xanthine treatment of apnea of prematurity. Pediatrics 55: 584 (1975). 7. Wesley-Hadzija, B., Mattocks, A. M.:
Specific thin-layer chromatographic method for the determination of theophylline in plasma in the presence of other drugs. J. Chromatogr. 115: 501 (1975). 8. Brockmann-Hanssen, E.,Olawuyi-Oke, T.: Gaschromatography of barbiturates, phenolic alkaloids and xanthine bases: Flashheater methypation by means of trimethylanilinium hydroxide. J. Pharm. Sci. 58: 370 (1969). 9. Thompson, R. D., Nagasawa, H. T., Jenne, J. W.: Determination of theophylline and
its metabolites in human urine and serum by high-pressure-liquid chromatography. J. Lab. Clin. Med. 84: 584 (1974). 10. Cook, G. L., Twine, M. E., Myers, M. et al.: Theophylline radioimmunassay. Res. Com. Chem. Pathol. Pharmacol. 13: 497 (1976). 11. Toxland, P. A., Oldigs, H. D.: The
determination of anticonvulsant drugs in dried whole blood samples on filter paper cards. Epileptology 1976 ed. D. Janz, publ. Thieme - Stuttgart, p. 416. 12. Albani, M., Toseland, P. A., Oldigs, H. D.: A new methodological approach to routine monitoring of anticonvulsant drugs. In preparation. 13. Bierman, C. W.: Theophylline in asthma. Pediatrics 58: 623 (1976).
Downloaded by: University of British Columbia. Copyrighted material.
selectivity of a nitrogen detector theophylline can now be readily chromatographed as the free compound in a crude ether extract of 35 mg whole blood on filter paper cards giving a result within about 20 minutes. The way of blood sampling has already been described for phenobarbitone (1 1) and has been made applicable in the meantime for the determination of several other anticonvulsant drugs (12). Now being used for more than three months the method has been proven advantageous for controlling theophylline drug levels in preterm infants.
