Br.J. Anaesth. (1978), 50, 143
A SENSITIVE GAS CHROMATOGRAPH ASSAY FOR THIOPENTONE IN PLASMA M. J. VAN HAMME AND M. M. GHONEIM SUMMARY
Thiopentone in biological fluids has been assayed most recently using spectrophotofluorimetry and a number of gas chromatograph methods. A recent pharmacokinetic study required the development of a flame ionization technique which would exhibit a sensitivity greater than that reported by Braddock and Marec (1965), yet circumvent the need for methylation as suggested by Becker (1976). It was important also that such an approach exhibit a wide range of linearity, similar to that reported by Dayton and others (1967). This communication describes an extraction procedure, coupled with a gas chromatograph analysis using a flame ionization detector, which permits determination of plasma concentrations of thiopentone in the 30.0-0.1 ;xg ml" 1 range, with a low-end sensitivity of less than 25 ng ml" 1 . METHODS
Linearity Human plasma was used to prepare standard solutions by diluting measured volumes of a freshly prepared aqueous stock solution of buffer-free sodium thiopentone 100 y.g ml" 1 (Lot 55-248-CA, Abbott Laboratories, North Chicago, Illinois, U.S.A.). The linearity of the 30.0-5.0 y.g ml" 1 concentration range was evaluated first by extracting 1.0-ml plasma aliquots, using butobarbitone acid 10.0 jxg (Ganes Chemical Works Inc., Carlstadt, New Jersey, U.S.A.) as internal standard. The 5.0-0.1 (xg ml" 1 range was examined similarly by extracting 2.0-ml plasma aliquots containing butobarbitone 2.5 fig. After duplicate gas chromatograph analysis of each sample, the linearity of each range was evaluated by a leastM. J. VAN HAMME, M.S., PH.D.; M. M. GHONEIM, M.B.,
B.CH., F.F.A.R.c.s.j Department of Anesthesia, University of Iowa College of Medicine, Iowa City, Iowa 52242, U.S.A.
squares regression analysis of the ratio of the peak heights of thiopentone to butobarbitone. Extraction procedure (1) Add 1.0 ml of an isopropyl alcohol solution of butobarbitone, followed by 1.5 mol litre" 1 monobasic sodium phosphate 3.0 ml and n-hexane 5.0 ml. (2) Shake with a horizontal shaker for 10 min, centrifuge at 10 min x 2000 rev min" 1 , quick-freeze the plasma layer in a dry ice/acetone bath, and transfer the organic phase to a 15-ml screw-capped centrifuge tube. (3) Add n-hexane 5.0 ml to the thawed, extracted plasma, and repeat step 2, centrifuging at 15x2000 rev min"1. After quick freezing, combine the nhexane extracts. (4) Add 2.5 mol litre" 1 sodium hydroxide 4.0 ml to each organic extract, shake and centrifuge as in step 2, and remove all n-hexane by aspiration. (5) Add n-hexane 3.0 ml to the basic aqueous extract, and shake, centrifuge and aspirate as in step 4. (6) Add 3.0 mol litre" 1 hydrochloric acid 4.0 ml, mix with a vortex mixer, and add methylene chloride 3.0 ml. Shake and centrifuge as in step 2, and aspirate the entire aqueous layer. (7) Carefully transfer the methylene chloride extract to an evaporating tube and place in a 40 °C water bath. Evaporate to dryness with a stream of dry nitrogen. Add carbon tetrachloride 10 [xlitre, mix with a vortex mixer, and inject 0.1-1.0 jxlitre onto the gas chromatograph column. Gas chromatography
A Bendix 2500 Research Gas Chromatograph equipped with a flame ionization detector was used for the analysis of plasma extracts. A 183 cm x 4 mm
Downloaded from http://bja.oxfordjournals.org/ at RMIT University Library on July 10, 2015
A sensitive flame ionization gas chromatograph assay method has been developed for thiopentone in human plasma. The extraction procedure minimizes desulphuration of the drug while removing most endogenous substances and is limited by a maximum of 50 ng ml" 1 in 2-ml plasma samples. There is linearity throughout the 30.0-5.0 (xg ml" 1 and 5.0-0.1 ng ml" 1 ranges. Extraction of six replicate 2.5-(ig ml" 1 samples resulted in a coefficient of variation of 3.0%. Similar treatment of six 0.20-[ig ml" 1 samples afforded a coefficient of variation of 4.6%.
BRITISH JOURNAL OF ANAESTHESIA
144 (i.d.) x 6 mm (o.d.) glass colunin was silanized with dimethyldichlorosilane (Sylon-CT, Supelco Inc., Bellefonte, Pennsylvania, U.S.A.) and packed with 5% OV-1 on 100/120 mesh HP Chromasorb W. It provided the desired barbiturate separations when operated at 205 °C, with the injection port and detector at 230 °C. The detector provided the optimum response when all gases were regulated at 207 kPa at the following flow rates: hydrogen 40 ml min" 1 ; air 425 ml min" 1 ; nitrogen, 45 ml min"1.
applicable; no specialized flame ionization detector is required as in the study of Sennello and Kohn (1974). The detector responses to prepared plasma solutions of thiopentone are shown in figure 2. The linearities exhibited throughout the combined 40
(1) ~
(2)
35
E
RESULTS AND DISCUSSION
(1.)
(2.)
(3.)
.2 25
1
| 20 o
A SENSITIVE GAS CHROMATOGRAPH ASSAY FOR THIOPENTONE IN PLASMA M. J. VAN HAMME AND M. M. GHONEIM SUMMARY
Thiopentone in biological fluids has been assayed most recently using spectrophotofluorimetry and a number of gas chromatograph methods. A recent pharmacokinetic study required the development of a flame ionization technique which would exhibit a sensitivity greater than that reported by Braddock and Marec (1965), yet circumvent the need for methylation as suggested by Becker (1976). It was important also that such an approach exhibit a wide range of linearity, similar to that reported by Dayton and others (1967). This communication describes an extraction procedure, coupled with a gas chromatograph analysis using a flame ionization detector, which permits determination of plasma concentrations of thiopentone in the 30.0-0.1 ;xg ml" 1 range, with a low-end sensitivity of less than 25 ng ml" 1 . METHODS
Linearity Human plasma was used to prepare standard solutions by diluting measured volumes of a freshly prepared aqueous stock solution of buffer-free sodium thiopentone 100 y.g ml" 1 (Lot 55-248-CA, Abbott Laboratories, North Chicago, Illinois, U.S.A.). The linearity of the 30.0-5.0 y.g ml" 1 concentration range was evaluated first by extracting 1.0-ml plasma aliquots, using butobarbitone acid 10.0 jxg (Ganes Chemical Works Inc., Carlstadt, New Jersey, U.S.A.) as internal standard. The 5.0-0.1 (xg ml" 1 range was examined similarly by extracting 2.0-ml plasma aliquots containing butobarbitone 2.5 fig. After duplicate gas chromatograph analysis of each sample, the linearity of each range was evaluated by a leastM. J. VAN HAMME, M.S., PH.D.; M. M. GHONEIM, M.B.,
B.CH., F.F.A.R.c.s.j Department of Anesthesia, University of Iowa College of Medicine, Iowa City, Iowa 52242, U.S.A.
squares regression analysis of the ratio of the peak heights of thiopentone to butobarbitone. Extraction procedure (1) Add 1.0 ml of an isopropyl alcohol solution of butobarbitone, followed by 1.5 mol litre" 1 monobasic sodium phosphate 3.0 ml and n-hexane 5.0 ml. (2) Shake with a horizontal shaker for 10 min, centrifuge at 10 min x 2000 rev min" 1 , quick-freeze the plasma layer in a dry ice/acetone bath, and transfer the organic phase to a 15-ml screw-capped centrifuge tube. (3) Add n-hexane 5.0 ml to the thawed, extracted plasma, and repeat step 2, centrifuging at 15x2000 rev min"1. After quick freezing, combine the nhexane extracts. (4) Add 2.5 mol litre" 1 sodium hydroxide 4.0 ml to each organic extract, shake and centrifuge as in step 2, and remove all n-hexane by aspiration. (5) Add n-hexane 3.0 ml to the basic aqueous extract, and shake, centrifuge and aspirate as in step 4. (6) Add 3.0 mol litre" 1 hydrochloric acid 4.0 ml, mix with a vortex mixer, and add methylene chloride 3.0 ml. Shake and centrifuge as in step 2, and aspirate the entire aqueous layer. (7) Carefully transfer the methylene chloride extract to an evaporating tube and place in a 40 °C water bath. Evaporate to dryness with a stream of dry nitrogen. Add carbon tetrachloride 10 [xlitre, mix with a vortex mixer, and inject 0.1-1.0 jxlitre onto the gas chromatograph column. Gas chromatography
A Bendix 2500 Research Gas Chromatograph equipped with a flame ionization detector was used for the analysis of plasma extracts. A 183 cm x 4 mm
Downloaded from http://bja.oxfordjournals.org/ at RMIT University Library on July 10, 2015
A sensitive flame ionization gas chromatograph assay method has been developed for thiopentone in human plasma. The extraction procedure minimizes desulphuration of the drug while removing most endogenous substances and is limited by a maximum of 50 ng ml" 1 in 2-ml plasma samples. There is linearity throughout the 30.0-5.0 (xg ml" 1 and 5.0-0.1 ng ml" 1 ranges. Extraction of six replicate 2.5-(ig ml" 1 samples resulted in a coefficient of variation of 3.0%. Similar treatment of six 0.20-[ig ml" 1 samples afforded a coefficient of variation of 4.6%.
BRITISH JOURNAL OF ANAESTHESIA
144 (i.d.) x 6 mm (o.d.) glass colunin was silanized with dimethyldichlorosilane (Sylon-CT, Supelco Inc., Bellefonte, Pennsylvania, U.S.A.) and packed with 5% OV-1 on 100/120 mesh HP Chromasorb W. It provided the desired barbiturate separations when operated at 205 °C, with the injection port and detector at 230 °C. The detector provided the optimum response when all gases were regulated at 207 kPa at the following flow rates: hydrogen 40 ml min" 1 ; air 425 ml min" 1 ; nitrogen, 45 ml min"1.
applicable; no specialized flame ionization detector is required as in the study of Sennello and Kohn (1974). The detector responses to prepared plasma solutions of thiopentone are shown in figure 2. The linearities exhibited throughout the combined 40
(1) ~
(2)
35
E
RESULTS AND DISCUSSION
(1.)
(2.)
(3.)
.2 25
1
| 20 o
