- Email: [email protected]
Biological AMS at Uppsala University: Status report
Nuclear Instruments and Methods in Physics Research B 268 (2010) 884–886
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Biological AMS at Uppsala University: Status report Mehran Salehpour *, Niklas Forsgard, Göran Possnert Ion Physics, Ångström Laboratory, Department of Engineering Sciences, Box 534, Uppsala University, SE-751 21 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Available online 7 October 2009 Keywords: AMS Biological Accelerator Mass Spectrometry DNA Microdosing
a b s t r a c t In January 2007 a new research program was initiated at Uppsala University focusing on the biological applications of AMS. We have used a 5 MV Pelletron Tandem accelerator to study biological samples. With Microdosing applications in mind, a variety of measurements have been performed on human blood, plasma and urine that have been labeled with a 14C-labeled pharmaceutical drug covering a concentration range, spanning 3 orders of magnitude. Furthermore, by studying small sample amounts and low concentrations, we have demonstrated sensitivity in the hundred zeptomole range for a small pharmaceutical substance in human blood. Another application of interest, based on the enhanced 14C activity from the cold war bomb-peak, is dating of DNA molecules providing fundamental data for the regenerative medicine and stem cell research community. We show data on a sensitive carrier method for measuring the isotopic ratio of small biological sample in the few lgC range. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction AMS was first implemented into the bio-analytical field by Vogel and coworkers [1]. Since then different isotopes have been used but the field is dominated by carbon-14 studies. A standard application involves a 14C-labeled molecule which is administered in the form of a pharmaceutical drug to animals or humans. The samples are taken as blood, urine, breath, tissue, biopsies, etc., and are analyzed with AMS. The availability of AMS and its ease of use have improved considerably during the last decade and consequently the expenditure associated with the technique has dropped substantially, reducing the cost of analyzing a sample significantly. This is partly as a result of the technology having become more mature and therefore more reliable, and partly due to the fact that new compact and less expensive accelerators have been introduced into the market. This has facilitated successful commercialization of AMS into the pharmaceutical and biochemical field. One exciting application of biological AMS in the pharmaceutical research and development sector is Microdosing [2] which has now become a major tool in drug development, where very small doses of drugs can be applied directly to humans at an early stage. Subsequently, the drug’s pharmacokinetic properties (e.g., absorption, distribution, metabolization and excretion) can be measured; the so called human phase-0 clinical trials. There has also been considerable interest for AMS within the stem cell and regenerative medicine research community [3–6]. The principle used is based on the release of large amounts of carbon-14 in the atmosphere * Corresponding author. Tel.: +46 18 471 3872; fax: +46 18 555 736. E-mail address: [email protected] (M. Salehpour). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.10.055
during the cold war era. The increased isotopic ratio (14C/12C) has been transcribed in the DNA molecules of the living organisms as a result of the ecological cycle. As the DNA molecule is stable and the chronological profile of the 14C to 12C ratio in living matter is well documented, AMS isotopic ratio measurement can be used to date the DNA samples with an accuracy of a few years. We here report on the status of the biological AMS activities at our laboratory which was initiated in the beginning of 2007. 2. Experimental There are two sample preparation laboratories on site. One is reserved for experiments with labeled samples of higher isotopic ratios (2–50 Modern) and the other for lower activity samples and carrier experiments. Different sample preparations methods are used depending on the type of sample as described below. 2.1. Method 1 When routine, high throughput sample production is required, we use the method of Ognibene et al. [7], which has been modified for preparation of small samples and will only be briefly described here. The samples (typically blood, plasma, urine) are placed in quartz tubes with CuO powder (100 mg), dried, evacuated and vacuum-sealed using a high temperature hydrogen and oxygen torch. The quartz tubes are then heated to 950 °C for 3 h and are allowed to cool slowly. The produced CO2 gas is transferred through a hypodermic needle into septa-sealed borosilicate vials (by cryogenic trapping) containing zinc (100 mg) and iron powder (2 mg) which is in a smaller vial, separated by a few borosilicate balls. The septa-seal vials are removed and heated to 530 °C for
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M. Salehpour et al. / Nuclear Instruments and Methods in Physics Research B 268 (2010) 884–886
6 h where the graphitization takes place as catalytic deposition of carbon (graphite) aggregates on the iron powder. The samples are finally collected and pressed into aluminium cathode targets (1 mm in diameter), placed in a holder and sent for AMS analysis. This method is not suited for low background application as it has a background of about 42,000 years. Nominally, about 1 mg of carbon is used for each sample corresponding to 10 lL of blood (10% carbon content), 25 lL plasma (4.5% carbon content), 100 lL of urine (about 1% carbon) or 3 mg of dried DNA (30% carbon), when available.
mated multiple sample holders. One ion source is home-made (25 samples) and is used for samples with high 14C contents. The other is a high throughput commercial ion source (40 samples, MC-SNICS, NEC) providing about 3 times higher current than the other ion source, used primarily for old archaeological samples which require low background.
2.2. Method 2
In an effort to characterize the dynamic range of our setup, we have used an anti-psychotic drug (Remoxipride, C16H23BrN2O3.HCl) as the labeled molecule for our experiments. The drug has been 14 C-marked with a specific activity of 2.035 GBq/mmol, dissolved in water and then in human blood. Starting from a concentration of about 170 pico-molar (activity of 0.36 Bq/mL) the sample was diluted in human blood in a number of steps spanning 3 orders of magnitude. The samples were prepared according to a modified version of method 1 [10], using total blood amounts which varied in volume from 0.1 to 10 lL. The total amount of drug per sample thus varied in the range from about 20 atto-grams to 3 pico-grams. The isotopic ratio is measured, and knowing the carbon mass of the sample, we can calculate the amount of labeled molecules per sample. Fig. 1 shows a plot of the measured number of moles of 14 C-Remoxipride in human blood versus the predicted values based on the dilution steps. A linear correlation exists over 4 orders of magnitude down to 170 zmole corresponding to 63 ag of Remoxipride. The variations between the measured and the expected values are partially due to inhomogeneous blood mixtures and are more pronounced at lower values. Another contribution to the scatter is that the sample preparation method is not optimal for the ultra-small samples, introducing analytical uncertainties.
4.1. Dynamic range data
4.2. Small samples We have measured the variation of the isotopic ratio with respect to the sample carbon mass for different samples [8,10,11], where measurements down to a few tens of lgC, can be performed. However, to extend the limit to lower masses (in the few lgC
Measured amount (zmole)
This is a modified version of method 1 above and is used when lower backgrounds are required (ca. 55,000 years) [8,9]. One major difference is that disposable plastics, such as tubes, connections, seals and valves, are not used in the setup and have been replaced with stainless steel. The reason for this choice is that the small samples are susceptible, specifically to carbon contaminants from miscellaneous plastic parts which are introduced during handling and gas transfer. The biological samples used in this method are often DNA or proteins in solution which are dried in vacuum and are then typically pumped with a turbo molecular pump for about half an hour. The larger liquid samples, often 1 mL solutions in water, are lyophilized to dryness deploying a cold trap. The samples are sealed and combusted as described in method 1. The quartz tubes are then connected to a vacuum system with the break-seal end into a metallic bellow which can be bent to puncture the break-seal and release the gas. After another 30 min the vacuum pump valve is closed and the sample gas is released and cryogenically transferred into a vial through a vacuum plug valve (Swagelok, Solon, Ohio, USA). After the gas transfer, the valve is closed, trapping the gas in the graphitization reactor consisting of a borosilicate vial (8 mm diameter, 40 mm) with 80 mg of zinc and a smaller vial containing iron powder (2 mg). The vial containing the sample gas is removed and placed in a heater block with the upper part of the vial, including the valve, remaining at room temperature. The sample is then graphitized and sent for AMS analysis. The carbon carrier method is implemented in cases where the amount of carbon in the sample is not sufficient to prepare graphite targets. Example are, the HPLC fractions from miscellaneous body fluids containing 14C-labeled drugs. As the carbon carrier, we have chosen a petrochemical compound Tributyrin (TRB, C15H26O6). The carbon-14 content varies significantly depending on the vendor by as much as a factor of 50. We have chosen Tributyrin from MP Biomedicals Inc. (Solon, Ohio, USA) with the lowest 14 C content (<500 zmol of 14C per mg 12C). Typically 1.5 mg of TRB corresponding to about of 1 mg of carbon per sample is used. The composite mixture of the carrier and the sample undergo the usual oxidation and reduction process to convert the sample into graphite according to method 2. In all cases, special care must be taken to avoid contamination by any carbon-containing substances or tools in the laboratory which could affect the isotopic ratio. All vials are pre-baked; 3 h at 950 °C for quartz vials and 6 h at 450 °C for borosilicate vials and parts.
4. Results and discussion
10
8
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1000
100
3. Accelerator Mass Spectrometry The experimental setup has been described in detail elsewhere [8–11] and is only outlined here. We have used the Uppsala University 5 MV Pelletron tandem accelerator (NEC Inc. Middleton, WI, USA) which was commissioned for use in 2001. Two independent cesium (Cs+) sputter ion sources are used, both with auto-
10 10
100
1000
10000
10
5
10
6
10
7
10
8
Predicted amount (zmole) Fig. 1. The measured total amount of 14C-Remoxipride in human blood per sample in zeptomole plotted against the predicted values.
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M. Salehpour et al. / Nuclear Instruments and Methods in Physics Research B 268 (2010) 884–886
in normal AMS. The advantage is the potential insensitivity to contamination since this is a constant amount added to each sample. To improve the method further, the amount of carbon in the sample needs to be known more accurately. Different DNA or protein samples may have somewhat different amounts of carbon which can affect the results. The method described in these proceedings by Zoppi et al., regarding the on-line total carbon measurement of the sample, could potentially improve the accuracy of this method. The measurements utilized the addition of a known mixture of 12C and 13C (6%) to a dead carbon carrier and measuring the 13C/12C and 14 13 C/ C and thus determining the total carbon amount.
8
Isotopic ratio (pMC)
7 6 5 4 3
4.3. HPLC fractions
2
We have prepared human plasma samples, containing a labeled drug in the femtoMolar range, i.e., concentrations where the sample isotopic ratio can not be resolved from the natural 14C-background using standard AMS. The plasma samples undergo protein precipitation followed by HPLC where the fraction containing the drug is separated. The fractions are added to a carrier as described above. We have been able to measure the concentration of a carbon-14 labeled drug in human plasma in the region below 20 fM [9] (1 fM = 1015 mol/L), and recently in the sub-fM region [14].
1 0 0
10
20
30
40
50
60
70
80
DNA carbon amount (µg) Fig. 2. The measured isotopic ratio of composite targets of carrier and sample in percent Modern (pMC) measured as a function of the amount of added carbon from herring sperm DNA. Two data seta are shown that were obtained under different experiments conditions.
5. Summary range) which is often required for the DNA work described earlier, we need a more sensitive technique. We are currently testing a new method inspired by the works of Khosh et al. [12] and Santos et al. [13]. Both methods use semi-empirical background subtraction schemes to compensate for the sample preparation’s background carbon contribution to the isotopic ratio. However, if the samples such as dilute biological compounds (e.g. DNA or proteins in water) introduce external carbon background, this would need to be accounted for. As an alternative approach, we have implemented a sensitive carrier method. Although the method is more time consuming, it can potentially eliminate an external carbon background [8] present in the sample as follows. The sample is added to a low 14C content carrier (<0.4 pMC) in steps of a few lgC. The isotopic ratios of the composite samples are subsequently measured. Fig. 2 shows the isotopic ratio, R, of the composite sample as a function of the amount of carbon, MDNA, in a herring sperm DNA sample for two sets of experiments, performed 6 months apart. As discussed in a recent paper [8], assuming that the contamination (lg) is much less than the mass of the composite sample (mg), the isotopic ratio of the DNA sample, RDNA, is given by
RDNA ¼ M carrier dR=dMDNA where Mcarrier is the amount of carbon in a carrier sample, R is the isotopic ratio of the composite sample and dR/dMDNA is the gradient of the plot in Fig. 2. Therefore, the isotopic ratio of the DNA sample can be deduced, knowing the mass of the carrier. It should be noted that the gradient is independent of an external carbon contamination [8].The R-value obtained from a linear fit is 102 ± 5 pMC, where the last point has been omitted as the amount of carbon from the sample (70 lg) starts to become significant compared to the carrier mass (1 mg) and consequently deviates form the straight line. The isotopic ratio obtained with standard AMS with a 1 mg DNA sample gives R = 101.2 ± 0.4 pMC. It is noted that error bar for the carrier method is significantly higher than standard AMS and needs to be improved. Nevertheless, in many instances in DNA dating measurements, such accuracies are sufficient to make this method analytically interesting. The disadvantage of the carrier method is the extra effort spent in making a few measurements instead of one
The biological AMS research program at Uppsala University consist of a number of activities including: (1) Microdosing measurements with 14C-labeled drugs in human/animal tissues, (2) Drug metabolite measurements using HPLC separation and carrier method and (3) Small sample AMS studies for, among others, age determination of DNA samples. Acknowledgements Uppsala BIO, Uppsala, Sweden, is gratefully acknowledged for the funding of the project and Kirsty Spalding and Jonas Frisén for providing the DNA samples. We also would like to thank Ira Palminge-Hallén (from the Swedish Medical Products Agency) and Lars Ståhle from AstraZeneca in Södertälje, for providing us with the labeled substance. References [1] J.S. Vogel, K.W. Turteltaub, TrAC Trends Anal. Chem. 11 (1992) 142. [2] G. Lappin, C. Garner, Nature Rev./Drug Discovery 2 (2003) 223. [3] R.D. Bhardwaj, M.A. Curtis, K.L. Spalding, B.A. Buchholz, D. Fink, T. BjörkEriksson, C. Nordborg, F.G. Gage, H. Druid, P.S. Eriksson, J. Frisén, Proc. Natl. Acad. Sci. 103 (2006) 12564. [4] K.L. Spalding, R.D. Bhardwaj, B.A. Buchholz, H. Druid, J. Frisén, Cell 122 (2005) 133. [5] K.L. Spalding, B.A. Buchholz, L.-E. Bergman, H. Druid, J. Frisén, Nature 437 (2005) 333. [6] K.L. Spalding, E. Arner, P.O. Westermark, S. Bernard, B.A. Buchholz, O. Bergmanm, L. Blomqvist, J. Hoffstedt, E. Näslund, T. Britton, H. Concha, M. Hassan, M. Rydén, J. Frisén, P. Arner, Nature 453 (2008) 783. [7] T.J. Ognibene, G. Bench, J.S. Vogel, Anal. Chem. 75 (2003) 2192. [8] M. Salehpour, N. Forsgard, G. Possnert, Rapid Commun. Mass Spectrom. 22 (2008) 3928. [9] M. Salehpour, N. Forsgard, G. Possnert, Rapid Commun. Mass Spectrom. 23 (2009) 557. [10] M. Salehpour, G. Possnert, H. Bryhni, Anal. Chem. 80 (2008) 3515. [11] M. Salehpour, G. Possnert, H. Bryhni, I. Palminger-Hallén, L. Ståhle, Appl. Radiat. Isotopes 67 (2009) 495. [12] M. Khosh, X. Xu, S.E. Trumbone, Nucl. Instr. and Meth. B 268 (2010) 927. [13] G.M. Santos, J.R. Southon, S. Griffin, S.R. Beaupre, E.R.M. Druffel, Nucl. Instrum. Meth. B 259 (2007) 293. [14] N. Forsgaard, M. Salehpour, G. Possnert. J. Anal. At. Spectrom., 2009, doi:10.1039/B906433H.
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Biological AMS at Uppsala University: Status report Mehran Salehpour *, Niklas Forsgard, Göran Possnert Ion Physics, Ångström Laboratory, Department of Engineering Sciences, Box 534, Uppsala University, SE-751 21 Uppsala, Sweden
a r t i c l e
i n f o
Article history: Available online 7 October 2009 Keywords: AMS Biological Accelerator Mass Spectrometry DNA Microdosing
a b s t r a c t In January 2007 a new research program was initiated at Uppsala University focusing on the biological applications of AMS. We have used a 5 MV Pelletron Tandem accelerator to study biological samples. With Microdosing applications in mind, a variety of measurements have been performed on human blood, plasma and urine that have been labeled with a 14C-labeled pharmaceutical drug covering a concentration range, spanning 3 orders of magnitude. Furthermore, by studying small sample amounts and low concentrations, we have demonstrated sensitivity in the hundred zeptomole range for a small pharmaceutical substance in human blood. Another application of interest, based on the enhanced 14C activity from the cold war bomb-peak, is dating of DNA molecules providing fundamental data for the regenerative medicine and stem cell research community. We show data on a sensitive carrier method for measuring the isotopic ratio of small biological sample in the few lgC range. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction AMS was first implemented into the bio-analytical field by Vogel and coworkers [1]. Since then different isotopes have been used but the field is dominated by carbon-14 studies. A standard application involves a 14C-labeled molecule which is administered in the form of a pharmaceutical drug to animals or humans. The samples are taken as blood, urine, breath, tissue, biopsies, etc., and are analyzed with AMS. The availability of AMS and its ease of use have improved considerably during the last decade and consequently the expenditure associated with the technique has dropped substantially, reducing the cost of analyzing a sample significantly. This is partly as a result of the technology having become more mature and therefore more reliable, and partly due to the fact that new compact and less expensive accelerators have been introduced into the market. This has facilitated successful commercialization of AMS into the pharmaceutical and biochemical field. One exciting application of biological AMS in the pharmaceutical research and development sector is Microdosing [2] which has now become a major tool in drug development, where very small doses of drugs can be applied directly to humans at an early stage. Subsequently, the drug’s pharmacokinetic properties (e.g., absorption, distribution, metabolization and excretion) can be measured; the so called human phase-0 clinical trials. There has also been considerable interest for AMS within the stem cell and regenerative medicine research community [3–6]. The principle used is based on the release of large amounts of carbon-14 in the atmosphere * Corresponding author. Tel.: +46 18 471 3872; fax: +46 18 555 736. E-mail address: [email protected] (M. Salehpour). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.10.055
during the cold war era. The increased isotopic ratio (14C/12C) has been transcribed in the DNA molecules of the living organisms as a result of the ecological cycle. As the DNA molecule is stable and the chronological profile of the 14C to 12C ratio in living matter is well documented, AMS isotopic ratio measurement can be used to date the DNA samples with an accuracy of a few years. We here report on the status of the biological AMS activities at our laboratory which was initiated in the beginning of 2007. 2. Experimental There are two sample preparation laboratories on site. One is reserved for experiments with labeled samples of higher isotopic ratios (2–50 Modern) and the other for lower activity samples and carrier experiments. Different sample preparations methods are used depending on the type of sample as described below. 2.1. Method 1 When routine, high throughput sample production is required, we use the method of Ognibene et al. [7], which has been modified for preparation of small samples and will only be briefly described here. The samples (typically blood, plasma, urine) are placed in quartz tubes with CuO powder (100 mg), dried, evacuated and vacuum-sealed using a high temperature hydrogen and oxygen torch. The quartz tubes are then heated to 950 °C for 3 h and are allowed to cool slowly. The produced CO2 gas is transferred through a hypodermic needle into septa-sealed borosilicate vials (by cryogenic trapping) containing zinc (100 mg) and iron powder (2 mg) which is in a smaller vial, separated by a few borosilicate balls. The septa-seal vials are removed and heated to 530 °C for
885
M. Salehpour et al. / Nuclear Instruments and Methods in Physics Research B 268 (2010) 884–886
6 h where the graphitization takes place as catalytic deposition of carbon (graphite) aggregates on the iron powder. The samples are finally collected and pressed into aluminium cathode targets (1 mm in diameter), placed in a holder and sent for AMS analysis. This method is not suited for low background application as it has a background of about 42,000 years. Nominally, about 1 mg of carbon is used for each sample corresponding to 10 lL of blood (10% carbon content), 25 lL plasma (4.5% carbon content), 100 lL of urine (about 1% carbon) or 3 mg of dried DNA (30% carbon), when available.
mated multiple sample holders. One ion source is home-made (25 samples) and is used for samples with high 14C contents. The other is a high throughput commercial ion source (40 samples, MC-SNICS, NEC) providing about 3 times higher current than the other ion source, used primarily for old archaeological samples which require low background.
2.2. Method 2
In an effort to characterize the dynamic range of our setup, we have used an anti-psychotic drug (Remoxipride, C16H23BrN2O3.HCl) as the labeled molecule for our experiments. The drug has been 14 C-marked with a specific activity of 2.035 GBq/mmol, dissolved in water and then in human blood. Starting from a concentration of about 170 pico-molar (activity of 0.36 Bq/mL) the sample was diluted in human blood in a number of steps spanning 3 orders of magnitude. The samples were prepared according to a modified version of method 1 [10], using total blood amounts which varied in volume from 0.1 to 10 lL. The total amount of drug per sample thus varied in the range from about 20 atto-grams to 3 pico-grams. The isotopic ratio is measured, and knowing the carbon mass of the sample, we can calculate the amount of labeled molecules per sample. Fig. 1 shows a plot of the measured number of moles of 14 C-Remoxipride in human blood versus the predicted values based on the dilution steps. A linear correlation exists over 4 orders of magnitude down to 170 zmole corresponding to 63 ag of Remoxipride. The variations between the measured and the expected values are partially due to inhomogeneous blood mixtures and are more pronounced at lower values. Another contribution to the scatter is that the sample preparation method is not optimal for the ultra-small samples, introducing analytical uncertainties.
4.1. Dynamic range data
4.2. Small samples We have measured the variation of the isotopic ratio with respect to the sample carbon mass for different samples [8,10,11], where measurements down to a few tens of lgC, can be performed. However, to extend the limit to lower masses (in the few lgC
Measured amount (zmole)
This is a modified version of method 1 above and is used when lower backgrounds are required (ca. 55,000 years) [8,9]. One major difference is that disposable plastics, such as tubes, connections, seals and valves, are not used in the setup and have been replaced with stainless steel. The reason for this choice is that the small samples are susceptible, specifically to carbon contaminants from miscellaneous plastic parts which are introduced during handling and gas transfer. The biological samples used in this method are often DNA or proteins in solution which are dried in vacuum and are then typically pumped with a turbo molecular pump for about half an hour. The larger liquid samples, often 1 mL solutions in water, are lyophilized to dryness deploying a cold trap. The samples are sealed and combusted as described in method 1. The quartz tubes are then connected to a vacuum system with the break-seal end into a metallic bellow which can be bent to puncture the break-seal and release the gas. After another 30 min the vacuum pump valve is closed and the sample gas is released and cryogenically transferred into a vial through a vacuum plug valve (Swagelok, Solon, Ohio, USA). After the gas transfer, the valve is closed, trapping the gas in the graphitization reactor consisting of a borosilicate vial (8 mm diameter, 40 mm) with 80 mg of zinc and a smaller vial containing iron powder (2 mg). The vial containing the sample gas is removed and placed in a heater block with the upper part of the vial, including the valve, remaining at room temperature. The sample is then graphitized and sent for AMS analysis. The carbon carrier method is implemented in cases where the amount of carbon in the sample is not sufficient to prepare graphite targets. Example are, the HPLC fractions from miscellaneous body fluids containing 14C-labeled drugs. As the carbon carrier, we have chosen a petrochemical compound Tributyrin (TRB, C15H26O6). The carbon-14 content varies significantly depending on the vendor by as much as a factor of 50. We have chosen Tributyrin from MP Biomedicals Inc. (Solon, Ohio, USA) with the lowest 14 C content (<500 zmol of 14C per mg 12C). Typically 1.5 mg of TRB corresponding to about of 1 mg of carbon per sample is used. The composite mixture of the carrier and the sample undergo the usual oxidation and reduction process to convert the sample into graphite according to method 2. In all cases, special care must be taken to avoid contamination by any carbon-containing substances or tools in the laboratory which could affect the isotopic ratio. All vials are pre-baked; 3 h at 950 °C for quartz vials and 6 h at 450 °C for borosilicate vials and parts.
4. Results and discussion
10
8
10
7
10
6
10
5
10000
1000
100
3. Accelerator Mass Spectrometry The experimental setup has been described in detail elsewhere [8–11] and is only outlined here. We have used the Uppsala University 5 MV Pelletron tandem accelerator (NEC Inc. Middleton, WI, USA) which was commissioned for use in 2001. Two independent cesium (Cs+) sputter ion sources are used, both with auto-
10 10
100
1000
10000
10
5
10
6
10
7
10
8
Predicted amount (zmole) Fig. 1. The measured total amount of 14C-Remoxipride in human blood per sample in zeptomole plotted against the predicted values.
886
M. Salehpour et al. / Nuclear Instruments and Methods in Physics Research B 268 (2010) 884–886
in normal AMS. The advantage is the potential insensitivity to contamination since this is a constant amount added to each sample. To improve the method further, the amount of carbon in the sample needs to be known more accurately. Different DNA or protein samples may have somewhat different amounts of carbon which can affect the results. The method described in these proceedings by Zoppi et al., regarding the on-line total carbon measurement of the sample, could potentially improve the accuracy of this method. The measurements utilized the addition of a known mixture of 12C and 13C (6%) to a dead carbon carrier and measuring the 13C/12C and 14 13 C/ C and thus determining the total carbon amount.
8
Isotopic ratio (pMC)
7 6 5 4 3
4.3. HPLC fractions
2
We have prepared human plasma samples, containing a labeled drug in the femtoMolar range, i.e., concentrations where the sample isotopic ratio can not be resolved from the natural 14C-background using standard AMS. The plasma samples undergo protein precipitation followed by HPLC where the fraction containing the drug is separated. The fractions are added to a carrier as described above. We have been able to measure the concentration of a carbon-14 labeled drug in human plasma in the region below 20 fM [9] (1 fM = 1015 mol/L), and recently in the sub-fM region [14].
1 0 0
10
20
30
40
50
60
70
80
DNA carbon amount (µg) Fig. 2. The measured isotopic ratio of composite targets of carrier and sample in percent Modern (pMC) measured as a function of the amount of added carbon from herring sperm DNA. Two data seta are shown that were obtained under different experiments conditions.
5. Summary range) which is often required for the DNA work described earlier, we need a more sensitive technique. We are currently testing a new method inspired by the works of Khosh et al. [12] and Santos et al. [13]. Both methods use semi-empirical background subtraction schemes to compensate for the sample preparation’s background carbon contribution to the isotopic ratio. However, if the samples such as dilute biological compounds (e.g. DNA or proteins in water) introduce external carbon background, this would need to be accounted for. As an alternative approach, we have implemented a sensitive carrier method. Although the method is more time consuming, it can potentially eliminate an external carbon background [8] present in the sample as follows. The sample is added to a low 14C content carrier (<0.4 pMC) in steps of a few lgC. The isotopic ratios of the composite samples are subsequently measured. Fig. 2 shows the isotopic ratio, R, of the composite sample as a function of the amount of carbon, MDNA, in a herring sperm DNA sample for two sets of experiments, performed 6 months apart. As discussed in a recent paper [8], assuming that the contamination (lg) is much less than the mass of the composite sample (mg), the isotopic ratio of the DNA sample, RDNA, is given by
RDNA ¼ M carrier dR=dMDNA where Mcarrier is the amount of carbon in a carrier sample, R is the isotopic ratio of the composite sample and dR/dMDNA is the gradient of the plot in Fig. 2. Therefore, the isotopic ratio of the DNA sample can be deduced, knowing the mass of the carrier. It should be noted that the gradient is independent of an external carbon contamination [8].The R-value obtained from a linear fit is 102 ± 5 pMC, where the last point has been omitted as the amount of carbon from the sample (70 lg) starts to become significant compared to the carrier mass (1 mg) and consequently deviates form the straight line. The isotopic ratio obtained with standard AMS with a 1 mg DNA sample gives R = 101.2 ± 0.4 pMC. It is noted that error bar for the carrier method is significantly higher than standard AMS and needs to be improved. Nevertheless, in many instances in DNA dating measurements, such accuracies are sufficient to make this method analytically interesting. The disadvantage of the carrier method is the extra effort spent in making a few measurements instead of one
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