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Analyst, February, 1975, VoZ. 100, pp. 99-104
99
A Modified Procedure for the Determination of Nicotine in Blood S. E. Falkrnan Research Laboratories, A B Leo, Helsingborg, Sweden
I, E. Burrows Department of Applied Research, Pedigree Petfoods Limited, Melton Mowbray, Leicestershire, L E I 3 1BB
R. A. Lundgren Research Laboratories, A B Leo, Helsingborg, Sweden
and B. F. J. Page Department of Metabolism and Pharmacokinetics, Huntingdon Research Centre, Huntingdon
An analytical method for the determination of nicotine has been established. It is a modification of an earlier reported method in which the over-all recovery of added nicotine has been increased from 55 to over 80 per cent. The determination is carried out by gas - liquid chromatography and the identity of the nicotine peak has been confirmed by mass spectrometry. The method has been applied to the determination of nicotine in the blood of cigarette smokers, and concentrations a t the level of 1 ng ml-1 can be measured.
For studies of human blood levels of nicotine, it is necessary to use a method of analysis that is sensitive enough to give accurate results at nicotine concentrations of a few nanograms per millilitre of blood. Most methods previously de~cribedl-~ are either difficult to apply or lack the required sensitivity for the determination of nicotine in this concentration range. Schievelbein and G r ~ n d k e ,Isaac ~ and Rand5 and Burrows et aLs have developed useful methods for the determination of small amounts of nicotine in blood. A modification of the gas-liquid chromatographic method developed by Burrows et aL6 is now reported. The purpose of our modifications has been to improve the over-all sensitivity of the methodas well as to decrease the level of interfering co-extractives.
Method Apparatus Steam-distillation apparatus. All glass. Varian 1400 gas - liquid chromatograph. Fitted with a flame-ionisation detector. LKB 2091 gas chromatograph - mass spectrometer combined with a n LKB 2130 data system. Reagents All reagents were of analytical-reagent grade. Water was double distilled from glass apparatus before use. Sodium hydroxide solution, 10 N. Alkaline salt solution. A solution of 300g of sodium hydroxide in 700ml of water, saturated with sodium chloride. Sulphuric acid, 1 N and 0-5 N. Dichloromethane. Benzene. Silicone M S antifoam A . Sodium chloride. 1-Nicotine. Obtained from Koch-Light Ltd., re-distilled under vacuum and stored at -10 "C under nitrogen. QuinoZine. Obtained from E. Merck A.G. Glass Equipment All glass equipment was treated overnight with Diversey Pyroneg cleaning fluid, rinsed several times with tap water and finally rinsed with ethanol before drying at 100 "C.
Published on 01 January 1975. Downloaded by University of Illinois at Chicago on 26/10/2014 17:04:08.
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FALKMAN et al.: A MODIFIED PROCEDURE FOR THE A d y s t , VoZ. 100 100 Procedure Obtain blood samples from the subjects by venepuncture in the arm. Dilute 10ml of heparinised whole blood with 10 ml of glass-distilled water, make the solution alkaline with 10 ml of alkaline salt solution and add 0.1 g of antifoam A. Steam distil the mixture at a distillation rate of 5 ml min-l, and collect the first 70 ml of distillate in a separating funnel containing 2 ml of 1 N sulphuric acid. Extract the acidified distillate with 10 ml of dichloromethane and discard the organic phase. Make the aqueous phase alkaline by adding 2 ml of 10 N sodium hydroxide solution and extract the mixture four times with 10 ml of dichloromethane. Extract the combined dichloromethane layers four times with 2 ml of 0.6 N sulphuric acid. Combine the aqueous layers in a 10-ml calibrated flask containing 2.5 g of sodium chloride and make the mixture alkaline with 1 ml of 10 N sodium hydroxide solution. Extract the nicotine with 100 p1 of benzene containing 5 pg ml-l of quinoline as an internal standard. Inject 6 p1 of the benzene solution on to the gas-chromatographic column using a Hamilton syringe.
Gas Chromatography
The gas chromatograph was fitted with a glass column (2 m x 3 mm id.) packed with 8 per cent. m/m Carbowax 20M and 2 per cent. m/m potassium hydroxide on acid-washed Chromosorb W, 80-100 mesh, treated with hexamethyldisilazane. The optimum chromatographic conditions for the determination of nicotine were as follows: column temperature, 150 "C ; nitrogen flow-rate, 27 ml min-1; hydrogen pressure, 2.2 x lo5 N m-2; air pressure, 4-1 x lo6N m-2; and nitrogen pressure, 3.1 x 105 N m-2. The retention time was about 6 min for nicotine and about 8 min for quinoline. At a signal to noise ratio of 3: 1, the detection limit for nicotine was 0.04 ng.
-
Gas Chromatography Mass Spectrometry
A glass column (2.7 m x 2 mm i.d.) packed with 6 per cent. m/m Carbowax 20M and 2 per cent. m/m potassium hydroxide was used. The gas-chromatographic operating conditions were as follows: column temperature, 170 "C; injection port temperature, 200 "C; helium flow-rate, 30 ml min-l; and inlet pressure, 3-1 x lo5 N m--2. The retention time for nicotine under these conditions was about 11 min. The mass-spectrometric operating conditions were as follows: ion-source temperature, 270 "C; inlet tube temperature, 230 "C; electron energy, 20 or 70 eV; trap current, 50 PA; accelerating voltage, 3-0 kV. After injection of a sample, a mass spectrum was scanned every third second. The spectra were stored on magnetic tape and, after the run, the total ion current was calculated by the computer and the total ion chromatogram was printed out together with mass chromatograms for three mass numbers typical for nicotine. This technique, ''mass chromatography," to detect the presence of a compound of known mass spectrum, was described by Hites and Biemann.' The mass spectrum of nicotine has four typical fragments (see, for example, McLafferty8 and SastryQ)at m/e 162 (molecular ion), 161, 133 and 84 (basic peak). In this investigation, the mass numbers 162, 133 and 84 were used in order to confirm the identity of extracted nicotine. The method required a little more substance than the standard procedure. A male volunteer smoked two cigarettes and 100 ml of blood were drawn after 5 min. The blood was divided into ten portions and analysed according to the standard procedure. The ten extracts were combined and the volume was reduced by extraction into sulphuric acid followed by re-extraction int.0 0.1 ml of benzene as described above. A 5-p1 volume was injected into the gas chromatography - mass spectrometry system, and chromatograms were obtained.
Results and Discussion Identification of Nicotine Chromatograms of extracts of blood from a smoker and of control blood are shown in Figs. 1 and 2. In order to establish the identity of nicotine extracted from a smoker's blood, the experiments with mass spectrometry were performed. As can be seen from the total ion chromatogram and the mass chromatograms in Fig. 3, all the typical fragments are present in the presumed nicotine peak. Mass spectra were printed at the beginning and end of the peak and both were identical with an authentic mass spectrum, thus proving that the sub-
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February, 1975
101
DETERMINATION OF NICOTINE I N BLOOD
Published on 01 January 1975. Downloaded by University of Illinois at Chicago on 26/10/2014 17:04:08.
stance extracted from blood was nicotine that had passed through the entire analytical procedure unchanged.
Fig. 1. Gas chromatogram of extract of blood from a smoker. 1, Nicotine; and 2, quinoline.
Fig. 2. Gas chromatogram of extract of control blood from a non-smoker. 2, Quinoline; and 3, blank.
As has also been observed by other analysts working on nicotine analysis (e.g., Isaac and Rand6), it was found that blood from non-smokers gave a small peak with the same retention time as nicotine. A repetition of the mass-spectrometric experiment using control blood from a non-smoker resulted in a mass chromatogram and mass spectra in which all the fragments characteristic of nicotine were present, thus indicating that nicotine may occur in small amounts also in the blood of non-smokers. This conclusion is in accordance with the discovery of Horning et aZ.10 that detectable amounts of nicotine are excreted in the urine of non-smokers who have been exposed to tobacco smoke. The blank value was determined and subtracted from the result obtained for every subject involved in the investigation, and had to be assumed to be constant during the time of the experiment. The method of analysis has been applied to several determinations of nicotine in the clinical field, the results of which will be published elsewhere, and the blank value mostly corresponded to about 5 per cent. of the internal standard or 3 ng ml-l in blood.
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84 V I
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Published on 01 January 1975. Downloaded by University of Illinois at Chicago on 26/10/2014 17:04:08.
Analyst, February, 1975, VoZ. 100, pp. 99-104
99
A Modified Procedure for the Determination of Nicotine in Blood S. E. Falkrnan Research Laboratories, A B Leo, Helsingborg, Sweden
I, E. Burrows Department of Applied Research, Pedigree Petfoods Limited, Melton Mowbray, Leicestershire, L E I 3 1BB
R. A. Lundgren Research Laboratories, A B Leo, Helsingborg, Sweden
and B. F. J. Page Department of Metabolism and Pharmacokinetics, Huntingdon Research Centre, Huntingdon
An analytical method for the determination of nicotine has been established. It is a modification of an earlier reported method in which the over-all recovery of added nicotine has been increased from 55 to over 80 per cent. The determination is carried out by gas - liquid chromatography and the identity of the nicotine peak has been confirmed by mass spectrometry. The method has been applied to the determination of nicotine in the blood of cigarette smokers, and concentrations a t the level of 1 ng ml-1 can be measured.
For studies of human blood levels of nicotine, it is necessary to use a method of analysis that is sensitive enough to give accurate results at nicotine concentrations of a few nanograms per millilitre of blood. Most methods previously de~cribedl-~ are either difficult to apply or lack the required sensitivity for the determination of nicotine in this concentration range. Schievelbein and G r ~ n d k e ,Isaac ~ and Rand5 and Burrows et aLs have developed useful methods for the determination of small amounts of nicotine in blood. A modification of the gas-liquid chromatographic method developed by Burrows et aL6 is now reported. The purpose of our modifications has been to improve the over-all sensitivity of the methodas well as to decrease the level of interfering co-extractives.
Method Apparatus Steam-distillation apparatus. All glass. Varian 1400 gas - liquid chromatograph. Fitted with a flame-ionisation detector. LKB 2091 gas chromatograph - mass spectrometer combined with a n LKB 2130 data system. Reagents All reagents were of analytical-reagent grade. Water was double distilled from glass apparatus before use. Sodium hydroxide solution, 10 N. Alkaline salt solution. A solution of 300g of sodium hydroxide in 700ml of water, saturated with sodium chloride. Sulphuric acid, 1 N and 0-5 N. Dichloromethane. Benzene. Silicone M S antifoam A . Sodium chloride. 1-Nicotine. Obtained from Koch-Light Ltd., re-distilled under vacuum and stored at -10 "C under nitrogen. QuinoZine. Obtained from E. Merck A.G. Glass Equipment All glass equipment was treated overnight with Diversey Pyroneg cleaning fluid, rinsed several times with tap water and finally rinsed with ethanol before drying at 100 "C.
Published on 01 January 1975. Downloaded by University of Illinois at Chicago on 26/10/2014 17:04:08.
View Article Online
FALKMAN et al.: A MODIFIED PROCEDURE FOR THE A d y s t , VoZ. 100 100 Procedure Obtain blood samples from the subjects by venepuncture in the arm. Dilute 10ml of heparinised whole blood with 10 ml of glass-distilled water, make the solution alkaline with 10 ml of alkaline salt solution and add 0.1 g of antifoam A. Steam distil the mixture at a distillation rate of 5 ml min-l, and collect the first 70 ml of distillate in a separating funnel containing 2 ml of 1 N sulphuric acid. Extract the acidified distillate with 10 ml of dichloromethane and discard the organic phase. Make the aqueous phase alkaline by adding 2 ml of 10 N sodium hydroxide solution and extract the mixture four times with 10 ml of dichloromethane. Extract the combined dichloromethane layers four times with 2 ml of 0.6 N sulphuric acid. Combine the aqueous layers in a 10-ml calibrated flask containing 2.5 g of sodium chloride and make the mixture alkaline with 1 ml of 10 N sodium hydroxide solution. Extract the nicotine with 100 p1 of benzene containing 5 pg ml-l of quinoline as an internal standard. Inject 6 p1 of the benzene solution on to the gas-chromatographic column using a Hamilton syringe.
Gas Chromatography
The gas chromatograph was fitted with a glass column (2 m x 3 mm id.) packed with 8 per cent. m/m Carbowax 20M and 2 per cent. m/m potassium hydroxide on acid-washed Chromosorb W, 80-100 mesh, treated with hexamethyldisilazane. The optimum chromatographic conditions for the determination of nicotine were as follows: column temperature, 150 "C ; nitrogen flow-rate, 27 ml min-1; hydrogen pressure, 2.2 x lo5 N m-2; air pressure, 4-1 x lo6N m-2; and nitrogen pressure, 3.1 x 105 N m-2. The retention time was about 6 min for nicotine and about 8 min for quinoline. At a signal to noise ratio of 3: 1, the detection limit for nicotine was 0.04 ng.
-
Gas Chromatography Mass Spectrometry
A glass column (2.7 m x 2 mm i.d.) packed with 6 per cent. m/m Carbowax 20M and 2 per cent. m/m potassium hydroxide was used. The gas-chromatographic operating conditions were as follows: column temperature, 170 "C; injection port temperature, 200 "C; helium flow-rate, 30 ml min-l; and inlet pressure, 3-1 x lo5 N m--2. The retention time for nicotine under these conditions was about 11 min. The mass-spectrometric operating conditions were as follows: ion-source temperature, 270 "C; inlet tube temperature, 230 "C; electron energy, 20 or 70 eV; trap current, 50 PA; accelerating voltage, 3-0 kV. After injection of a sample, a mass spectrum was scanned every third second. The spectra were stored on magnetic tape and, after the run, the total ion current was calculated by the computer and the total ion chromatogram was printed out together with mass chromatograms for three mass numbers typical for nicotine. This technique, ''mass chromatography," to detect the presence of a compound of known mass spectrum, was described by Hites and Biemann.' The mass spectrum of nicotine has four typical fragments (see, for example, McLafferty8 and SastryQ)at m/e 162 (molecular ion), 161, 133 and 84 (basic peak). In this investigation, the mass numbers 162, 133 and 84 were used in order to confirm the identity of extracted nicotine. The method required a little more substance than the standard procedure. A male volunteer smoked two cigarettes and 100 ml of blood were drawn after 5 min. The blood was divided into ten portions and analysed according to the standard procedure. The ten extracts were combined and the volume was reduced by extraction into sulphuric acid followed by re-extraction int.0 0.1 ml of benzene as described above. A 5-p1 volume was injected into the gas chromatography - mass spectrometry system, and chromatograms were obtained.
Results and Discussion Identification of Nicotine Chromatograms of extracts of blood from a smoker and of control blood are shown in Figs. 1 and 2. In order to establish the identity of nicotine extracted from a smoker's blood, the experiments with mass spectrometry were performed. As can be seen from the total ion chromatogram and the mass chromatograms in Fig. 3, all the typical fragments are present in the presumed nicotine peak. Mass spectra were printed at the beginning and end of the peak and both were identical with an authentic mass spectrum, thus proving that the sub-
View Article Online
February, 1975
101
DETERMINATION OF NICOTINE I N BLOOD
Published on 01 January 1975. Downloaded by University of Illinois at Chicago on 26/10/2014 17:04:08.
stance extracted from blood was nicotine that had passed through the entire analytical procedure unchanged.
Fig. 1. Gas chromatogram of extract of blood from a smoker. 1, Nicotine; and 2, quinoline.
Fig. 2. Gas chromatogram of extract of control blood from a non-smoker. 2, Quinoline; and 3, blank.
As has also been observed by other analysts working on nicotine analysis (e.g., Isaac and Rand6), it was found that blood from non-smokers gave a small peak with the same retention time as nicotine. A repetition of the mass-spectrometric experiment using control blood from a non-smoker resulted in a mass chromatogram and mass spectra in which all the fragments characteristic of nicotine were present, thus indicating that nicotine may occur in small amounts also in the blood of non-smokers. This conclusion is in accordance with the discovery of Horning et aZ.10 that detectable amounts of nicotine are excreted in the urine of non-smokers who have been exposed to tobacco smoke. The blank value was determined and subtracted from the result obtained for every subject involved in the investigation, and had to be assumed to be constant during the time of the experiment. The method of analysis has been applied to several determinations of nicotine in the clinical field, the results of which will be published elsewhere, and the blank value mostly corresponded to about 5 per cent. of the internal standard or 3 ng ml-l in blood.
I
1
84 V I
0
11
I
2 2 3
I
34
t
54
I
I
6 5 7
68
97
I
18 0
I
I
91 1 1 0
