Measurement of total boron and 10B concentration and the detection and measurement of elevated 10B levels in biological samples by inductively coupled plasma mass spectrometry using the determination of 10B511B ratios Jennifer A. Moretona and H. Trevor Delvesb aMilestones, Ampleforth, North Yorkshire, UK YO624DA bSAS Trace Element Unit, Chemical Pathology, Southampton General Hospital, Southampton, UK SO16 6YD Received 19th April 1999, Accepted 21st July 1999 Methods were developed to determine total B and 10B content in various biological samples, media and reagents by ICP-MS.Open vessel, wet ash digestion protocols using HNO3 were developed for the biological and media samples. Digestion of blood and plasma also required H2SO4. Despite relatively high blanks, instrumental detection limits using 3s at or near the blank level were, for total boron (total B), <0.1 mmol l-1 (<1 mg l-1), and for 10B, <0.01 mmol l-1 (<0.1 mg l-1). Problems with variable blanks, matrix suppression and peak overlap by 12C on 11B are discussed.The developed methods were used to analyse samples for a collaborative study with Clinical Neurosciences at Southampton General Hospital, related to boron neutron capture therapy of brain tumours, of the uptake of 10B by tumour cells in vitro, using tissue culture, and in vivo, in the rat. Detection limits achieved in cells solubilised in 1 MNaOH and medium were<0.001 mmol ml-1 total B and<0.0001 mmol ml-1 10B and, for blood, 0.002 mmol g-1 total B and 0.0002 mmol g-1 10B,~10-9 Mboronophenylalanine solutions.The analytical precision for duplicate analyses gave mean within-run RSDs of ~7% for 10B concentration and <10% for total B concentration. The determination of 10B511B ratios enabled statistically significant increases in 10B concentration to be detected and confirmed at low B levels where sample digests and digest blanks had similar B contents. Running 10B511B ratios for standard and sample solutions were utilised for novel methods of calculating elevated 10B levels in the samples.Despite achieving low detection limits, elevated 10B levels could not be confirmed without the determination of 10B511B ratios. Boron neutron capture therapy (BNCT) can be used to treat to BNCT, finding a good correlation between determination by ICP-MS and ICP-AES, which has also been found by other brain tumours for which tumour cells are targeted via the incorporation of 10B.Boron has two isotopes, 11B (80.22%) workers.35 ICP methods give the best precision and lower detection limits.34 ICP-MS has the advantages of a sensitivity and 10B (19.78%).1 The neutron capture reaction for 10B is 10B(n,ac)7Li. The boron-containing substances used for BNCT about an order of magnitude higher than ICP-AES and discrimination between the two isotopes. are 10B-enriched. The production of 10B-enriched boron only began in the 1940s.2 Analysis for B was carried out in the Trace Element Unit as part of a collaborative study with Clinical Neurosciences For successful BNCT, suYcient 10B atoms are required in the cancer cells and suYcient neutrons must be delivered to at Southampton General Hospital.The research group of Mr P. D. Lees and Dr. T. Loughlin was working on the uptake the tumour, related to tumour location and depth.3 The most critical and diYcult step is targeting the tumour. A high of 10B by tumour cells in vitro, using tissue culture, and in vivo, in the rat, including the uptake of specific 10B-enriched concentration diVerential is required for 10B between the normal and neoplastic cells.Suitable 10B concentrations in peptides. Methods were developed to determine total B and 10B content by ICP-MS, enabling uptake of 10B by cells to be tumour cells proposed were >10 mg g-1 (ref. 2) and 35–50 mg g-1 (ref. 4). Early experiments with BNCT utilised assessed. Biological samples included blood, plasma, brain tissue, brain homogenate, cells and cells solubilised in 1 M boric acid,2 borax5 and sodium pentaborate.6 Then p-carboxyphenylboronic acid6 and derivatives of polyhedral boranes NaOH.Incubation media and wash media used for cells were also analysed to check on B levels and washout. Other reagents were used.7,8 p-Boronophenylalanine (BPA) gave encouraging results with malignant melanomas.9–16 Compounds now under were also analysed to check for B contamination.Samples of boric acid (H3BO3), BPA [H2BO2C6H4CH2CH(NH2)COOH] evaluation include carborane-containing amino acids,17 boronated thioureas and boron compounds conjugated to and carboranylacetic acid (COOHCH2–C2B10H11) were also analysed, in part to verify that they were enriched with 10B. tumour-seeking macromolecules,18 such as monoclonal antibodies, 19,20 nucleosides, oligonucleotides and polypeptides. Open vessel, wet ash digestion protocols using HNO3 were developed for the biological and media samples.Digestion The ‘prompt gamma’ technique21 was the first available method to measure 10B levels in patient’s blood in 1986, since was diYcult and time consuming and the optimum conditions varied for each type of sample. Digestion of blood and plasma when other methods have been introduced.22–27 By the early 1990s, most NCT teams were using inductively coupled plasma also required H2SO4. Published reports comparing sample dissolution methods for B, including dry ashing, wet digestion atomic emission spectrometry (ICP-AES).28,29 B levels have been determined in biological reference materials by ICP- and microwave dissolution, have produced contradictory results.35 A study by Nyomura et al.35 found no B loss during MS30–35 but have only recently been used by NCT teams.36 Probst et al.36 determined B in mouse tissue for a study related open vessel digestion.J. Anal. At. Spectrom., 1999, 14, 1545–1556 1545Initial experiments at the Trace Element Unit yielded tissue Isotopic shifts therefore occur in terrestrial rocks.30 The 10B511B ratio of terrestrial samples varies from 0.236 to containing 100 mg g-1 10B, but the high cost of the 10Bcontaining compounds was an incentive to work at relatively 0.25537–39 and a ratio of 0.260 has been reported for meteoritic material.30 TIMS has been almost exclusively used in B isotope low concentrations, where problems of contamination and high B digestion blanks became significant.EVorts were made geochemistry40 but one investigation, using ICP-MS, showed that the 10B511B ratio was 0.256 for basalt and 0.239 for to keep background concentrations as low as possible (<5 mg l-1 total B). In order to achieve adequate detection seaweed.38 limits, a sensitivity for total B of >75 000 counts s-1 with a 100 mg l-1 B solution was required. Experimental Despite relatively high background concentrations, instrumental detection limits (DLs) (using three times the standard Instrumental conditions deviation of replicate determinations made at or near the blank level ) were <0.1 mmol l-1 (<1 mg l-1) for total B and Initial investigations were carried out using a VG ( Winsford, Cheshire, UK) PlasmaQuad Mark 1 instrument but in the <0.01 mmol l-1 (<0.1 mg l-1) for 10B.The calculated sample DLs for B (10B, 11B and total B) in biological samples were majority of the analyses a Perkin-Elmer (PE) SCIEX ( Thornhill, ON, Canada) Elan 5000 was used.Details of the as low as 0.001 mmol g-1 (0.01 mg g-1) (10-9M boronophenylalanine solutions), but there were frequent problems with the operating conditions used with the two instruments are given in Table 1. The scan parameters diVered, since the multi- variable digest blanks, background B correction and matrix suppression. These problems have been experienced by other channel scanning mode was used for analysis with the VG PlasmaQuad and the peak hopping mode with the PE SCIEX workers.30,35,36 10B511B ratios were monitored from the beginning of the Elan 5000.Sample preparation procedures were identical for the two instruments. study and it became clear that increases in sample 10B level could be detected using the 10B511B ratio, below the calculated When operating in the multi-channel scanning mode, any overlap of the 12C peak on the 11B peak was visible, necessitat- DL for total B or 10B. 10B511B ratio determination was suYciently precise to enable statistically significant increases ing further digestion of the samples. Peak tailing eVects resulting from overlap of the 12C peak on the 11B peak with in 10B concentration to be detected and confirmed at these low levels. Running 10B511B ratios for standard and sample organic samples have been identified by other workers.33–36,41 For the PE SCIEX Elan 5000, operating in the peak hopping solutions were utilised for novel methods of calculating elevated 10B levels in the samples.The diVerence in m/z between mode, there was less indication of C levels in the samples. An initial 1 min narrow mass range scan (Table 2, boron scan) 10B and 11B is relatively large, therefore micromolar quantities were used for calculations. Despite achieving low DLs, elevated was therefore carried out with the PE SCIEX Elan 5000 in the single continuous scan mode, in order to check C levels 10B levels could not be confirmed without the determination of 10B511B ratios.and also find out whether sample dilution was necessary before analysis. The 10B511B ratio using the normal abundance of the two isotopes of boron is 0.2466.1 For the purpose of this study, For the VG PlasmaQuad a narrow mass range scan of 5–11.5 u for 2 min was used, which included the two B peaks, the background B present was assumed to have the normal abundance for the two isotopes. Boron is known to form 10B and 11B, and one or both of the two internal standards, lithium (7Li) and beryllium (9Be) (Table 2, boron content). volatile compounds and is mobile in the natural setting.Table 1 Operating conditions for ICP-MS instruments VG PlasmaQuad Mark 1— Torch box VG Model 765 Rf generator (ICP) Henry electronics Rf incident power 1370 W Reflected power 5 W (usually 2–3W) Nebuliser Meinhard concentric Type A (TR-30-A3) Spray chamber Scott, double-pass Sample solution uptake rate (Gilson, Minipuls) 0.8 ml min-1 Argon plasma gas flow rate 14.5 l min-1 Argon auxiliary gas flow rate 0.8 l min-1 Argon nebuliser gas flow rate 0.76 l min-1 Sample cone Nickel, aperture 1 mm Skimmer cone Nickel, aperture 0.75 mm Load coil–sampling cone distance 10 mm Electron multiplier Continuous dynode ion multiplier (Channeltron, Galileo) Multichannel analyser TN7200 (Tracor Northern) Data system Compaq 386/20e computer Gas flows and torch position were optimised for maximum sensitivity Perkin-Elmer SCIEX Elan 5000— Rf incident power 1010 W Nebuliser Ryton, crossflow Spray chamber Ryton with flow spoiler Argon plasma gas flow rate 15.0 l min-1 Argon auxiliary gas flow rate 0.8 l min-1 Argon nebuliser gas flow rate 0.9 l min-1 Sample cone Nickel, aperture 1.1 mm Skimmer cone Nickel, aperture 0.9 mm Load coil–sampling cone distance 10 mm Electron multiplier Continuous dynode ion multiplier (Channeltron, Galileo) Data system IBM Personal System/2 Model 70 386 computer All other conditions were standard Gas flows were optimised for maximum sensitivity 1546 J.Anal. At. Spectrom., 1999, 14, 1545–1556Table 2 Scan parameters for boron determination for Perkin-Elmer SCIEX Elan 5000 and VG PlasmaQuad Mark 1 instruments Boron content Boron scan: PE SCIEX Elan VG PlasmaQuad (~1 min narrow (~2 min narrow 10B511B determination: Parameter mass range scan) mass range scan) PE SCIEX Elan PE SCIEX Elan Scanning mode Single continuous Multi-channel Peak hopping Peak hopping scan Mass range (u) 4–13 5–11.5 7, 9, 10 and 11 9, 10 and 11 No.of channels 100 512 4 3 Dwell time/ms 100 0.16 80 80 No. of sweeps 6 1500 75 143 Acquisition/s 62 122 25 36 No. of replicates 5 5 Total acquisition time/s 126 180 Similarly, 2 min scans (Table 2, boron content) in the peak the second internal standard for analysis. Since the degree of ionisation of Be is only 75%,42 a higher concentration was hopping mode using the 10B, 11B and one or both of the two internal standard peaks, 7Li and 9Be, were adequate for used (30 mg l-1 Be).For later analysis where B levels were known to be near the detection limit, Be was used as the determining the relatively high B concentrations at the beginning of the study with the PE SCIEX Elan 5000. Later, for internal standard, since contamination with Li was more likely. samples with very low 10B concentrations, where accurate 10B511B ratio determination was essential, the maximum acqui- Sample digestion/preparation sition time (3 min) was utilised for the sample volume available Two basic protocols were developed for sample digestion: the and only one internal standard peak, 9Be in this case (Table 2, first was for blood and plasma and the second was for other 10B511B ratio determination).biological samples including brain tissue, brain homogenate, The normal resolution mode was used for all analyses cells, cells solubilised in 1 M NaOH, incubation medium and (resolution set at 0.8 u at 10% peak height), since with high wash medium. There were variations with the second method resolution (resolution set at 0.6 u at 10% peak height) not depending on the type of sample, which aVected the ease of only the intensities but also the precision for isotope ratio digestion. A two- or three-stage digestion procedure was determination were reduced. utilised in part to reduce C levels in the final digest.The 12C peak could overlap the 11B peak if high C levels were present.Reagents The reduction of the C content in digests also helped to The reagents used were of the highest purity available unless prevent blockage of the torch and nebuliser and deposition on stated otherwise (Aristar or AnalaR grade from BDH, Poole, the cones, enabling long runs to be utilised. Dorset, UK, or their equivalents from Johnson Matthey, Where appropriate, samples were pipetted into quartz flasks Royston, Hertfordshire, UK).De-ionised water was provided for digestion. Biological samples were weighed accurately by by a Milli-Q system (Millipore, Watford, UK). diVerence either using the container in which the sample Stock mono-elemental standard solutions at 1000 mg l-1 arrived or the quartz flask used for digestion. Samples were boron (BDH, Spectrosol ) and 10 000 mg l-1 lithium and beryl- only analysed in duplicate if suYcient was available, which in lium (Johnson Matthey, Specpure) were used to prepare sub- practice only applied to a limited number of samples of blood, stock solutions at 10 and 100 mg l-1, containing no acid.plasma and media and one batch of cells solubilised in 1 M Working standard solutions of 1 mg l-1 B, 1 mg l-1 Li and NaOH. For blood and plasma, duplicate samples were pre- 5 mg l-1 Be containing no acid were used for final dilutions. pared if the total mass of the sample was >0.5 g and for The normal wash solution used for ICP-MS was 1% v/v media and solubilised cells if the volume of the sample was HNO3–0.1% v/v Triton X-100 solution, which was prepared >1 ml.The biological sample size range, except the one batch by diluting 10 ml of HNO3 and 1 ml of Triton X-100 to 1 l of solubilised cells, was 48–934 mg [Nyomura et al.35 recwith de-ionised water. De-ionised water was also used. ommended a sample size of 0.5 g, particularly for samples A 5%v/v Triton X-100 solution was prepared by diluting with a low B concentration (<3 mg g-1)]. 50 ml of Triton X-100 to 1 l with de-ionised water. At least three blanks were utilised, one with no internal standard, and treated identically with the samples but replacing Methods the sample with the same volume of de-ionised water. A further blank was added if there was a likelihood of the All standard solutions were prepared and final sample dilutions presence of high levels of B requiring sample dilution. were carried out in 30 ml polycarbonate containers fitted with screw caps (Universals, Sterilin, Richmond, Surrey, UK). Where possible, samples were analysed in duplicate.Whole blood and plasma. Approximately 0.5 g of blood or plasma (range 0.17–1.2 g) was weighed accurately by diVerence Initially an internal standard of 10 mg l-1 Li was used. Beryllium was also a satisfactory internal standard and was and placed in a 25 ml quartz conical flask. The internal standard, 100 ml of a 1mg l-1 Li solution or 60 ml of a 5mg l-1 used for blood samples for which lithium heparin had been added as an anticoagulant.The internal standard was added Be solution, was added, then 1–2 ml of de-ionised water, 1 ml of concentrated HNO3 (16M) and 100 ml of concentrated before acid digestion of the samples. For some digested samples, the B concentration was too high (>100 mg l-1) for H2SO4 (36 M). The contents of the flask were then evaporated on a hot-plate (150 °C) until fumes of H2SO4 were produced.analysis without further dilution. In these cases, since an internal standard had already been added before digestion, After cooling for ~10 min, a further 1 ml of concentrated HNO3 was added to each flask and evaporated to dryness the alternative internal standard was added on sample dilution for analysis; to avoid confusion, 10 mg l-1 Li was added as the (with slight charring). After cooling, 0.2 ml of concentrated HNO3 was added to each flask and warmed gently on the hot- first internal standard for digestion and 30 mg l-1 Be as J.Anal. At. Spectrom., 1999, 14, 1545–1556 1547plate, then 2–3 ml of de-ionised water and finally 0.3 ml of The BPA solutions were provided in 1 M NaOH. For the lowest dilution utilised (1+1.7), 100 ml concentrated HNO3 concentrated HNO3 to dissolve the residue. The flasks were removed from the hot-plate and allowed to cool for several were added to a total solution volume of 2.7 ml to neutralise the alkali.hours or overnight. The resulting solutions were then transferred quantitatively into weighed polycarbonate containers Aqueous calibrating standards and made up to 10 g by weight, using 4–6 washings with de-ionised water. Owing to the variety of types of sample analysed, aqueous standards were utilised. Brain tissue, brain homogenate and cells. The sample prep- A series of aqueous calibrating standards for B were pre- aration was as described for the analysis of blood and plasma pared in polycarbonate containers at 0, 5, 10 and 20 mg l-1 but there was no addition of concentrated H2SO4.The sample (occasionally also 40 and 80 mg l-1) containing either 10 mg l-1 size was usually smaller, generally 100 mg (range Li or 30 mg l-1 Be as an internal standard. Working standard 33–460 mg). solutions of 1 mg l-1 B, 1 mg l-1 Li and 5 mg l-1 Be were used. Standards with a total volume of 10 ml were prepared Solubilised cells. Digestion of cells solubilised in 1M NaOH by pipetting 0, 50, 100, 200 ml (400 and 800 ml ) of the 1 mg l-1 was more diYcult than that of non-solubilised cells, requiring B solution into polycarbonate containers.If the internal stana three-stage procedure. dard was Li, then 9.90, 9.85, 9.80, 9.70 ml (9.50 and 9.10 ml ) Samples of solubilised cells of known volume (5 ml ) were of de-ionised water were added to each container, respectively, transferred into 25 ml quartz flasks with three washes of followed by 100 ml of 1mg l-1 Li solution to all the containers.de-ionised water. The internal standard, 100 ml of 1mg l-1 Li If the internal standard was Be, then 9.94, 9.89, 9.84, 9.74 ml solution or 60 ml of 5mgl-1 Be solution, was added, then (9.54 and 9.14 ml ) of de-ionised water was added to each 1 ml of concentrated HNO3, and the contents were carefully container, respectively, followed by 60 ml of 5mg l-1 Be solu- evaporated to dryness on a hot-plate (150 °C), to avoid spitting tion to all the containers.(heavy residue). After cooling, a further 1 ml of concentrated HNO3 was added to each flask and evaporated to dryness Results and discussion (residue slightly reduced). Then, after cooling, a further 1 ml of concentrated HNO3 was added to each flask and evaporated Washout for boron to dryness (residue reduced). After cooling, 0.5 ml of concentrated HNO3 was added to each flask and warmed gently on In general, the wash utilised between samples was 1 min with the normal wash solution (1% v/v HNO3–0.1% v/v Triton the hot-plate, then 2–3 ml of de-ionised water with gentle warming (residue dissolved).The flasks were removed from X-100 solution) followed by 3 min with fresh de-ionised water so an automatic sampler could not be used. Before a run of the hot-plate and allowed to cool for several hours or overnight. The resulting solutions were transferred into weighed samples was initiated, a 3 min wash with the normal wash solution followed by a 6 min wash with fresh de-ionised water polycarbonate containers and made up to 10 g by weight using 4–6 washings with de-ionised water.was used, in order to minimise background B levels. To reduce run times, standard solutions were run in order of ascending concentration utilising a wash of 1 min with de-ionised water Media. Media samples also required a three-stage digestion procedure. between samples. For samples known to contain low B levels from the initial rapid 1 min scan, the wash with 1% v/v A 1 ml volume of medium and 2 ml of de-ionised water were placed in a 25 ml quartz flask.The internal standard, HNO3–0.1% v/v Triton X-100 solution was omitted, since the wash itself contained low levels of B, and replaced by a wash 100 ml of 1mg l-1 Li solution or 60 ml of 5mg l-1 Be solution, was added, then 1 ml of concentrated HNO3, and the contents of 3 min with fresh de-ionised water. Memory eVects are thought to be caused by the release of were carefully evaporated to dryness on a hot-plate (150 °C), to avoid frothing (frothy yellow residue).After cooling, 0.5 ml adhered B from the instrument to subsequent samples and B volatilisation in the spray chamber.35 Since preliminary scans of concentrated HNO3 was added to each flask and evaporated to dryness (residue slightly reduced). After cooling, a further had established the approximate B concentration in each sample, care was taken to run samples in ascending order of 0.5 ml of concentrated HNO3 was added to each flask and evaporated to dryness (fine yellow residue).After cooling, concentration and check on background levels after running high standards, unexpectedly high samples and occasionally 0.2 ml of concentrated HNO3 was added to each flask and warmed gently on the hot-plate, then 2–3 ml of de-ionised during the run. There was a tendency for background B counts s-1 to slowly water with gentle warming (the residue should dissolve).The flasks were removed from the hot-plate and allowed to cool fall during runs. This eVect was more pronounced with the VG PlasmaQuad where the background concentration could for several hours or overnight. The resulting solutions were transferred into weighed polycarbonate containers and made fall by the equivalent of 1 mg l-1 total B per hour. Wash solutions were easily contaminated by B. The utilis- up to 10 g by weight using 4–6 washings with de-ionised water.ation of fresh wash solutions was essential. The Perkin-Elmer AS-90 autosampler did not have a continuous rinse facility Preparation of samples not requiring digestion. Other reagents used by Clinical Neurosciences were analysed to check for B and the wash container was relatively small, ~250 ml, increasing the likelihood of cross-contamination. This was particularly contamination, including saline solution, TRIS buVer, Evan’s Blue solution and lithium heparin solution.The volumes important when samples containing very variable quantities of B were run (range <1–100 mg l-1). The VG PlasmaQuad provided varied from 0.2 to 1.1 ml and were made up to 10 ml with de-ionised water in polycarbonate containers and spiked autosampler was a Gilson Model 221 with a continuous rinse facility, but the PlasmaQuad was only used at the beginning with an internal standard, usually 30 mg l-1 Be, for analysis by ICP-MS. of the study.The 0.5% v/v Triton X-100 solution contained up to The B-containing compounds analysed were provided in solution, including boric acid, o-carboranylacetic acid and p- 1mg l-1 total B and the 2% v/v HNO3 solution contained up to 2.5 mg l-1 total B. Solutions containing ammonia were boronophenylalanine (BPA). Suitable dilutions in polycarbonate containers using de-ionised water were made, spiked with found to contain very high levels of B (up to 100 mg l-1), which precluded the direct dilution of blood solutions for 10 mg l-1 Li or 30 mg l-1 Be as an internal standard. 1548 J. Anal. At. Spectrom., 1999, 14, 1545–1556analysis, since ammonia was present in the normal diluent and to fall during long runs, as shown by the second aqueous calibration curve, possibly related to matrix eVects. wash solutions used for blood.43 Despite the presence of B in HNO3, the high acid content Although Be has consistently been reported to be the best internal standard to use for B determination by of the sample digests (2–5% HNO3) appeared to aid washout and reduce background levels.Alternatively, this could have ICP-MS,33–35,44 several research groups have found that HNO3 suppresses the Be signal more than the B signal.34,35 However, been caused by matrix eVects such as a memory eVect giving rise to matrix suppression.41 this would not explain the non-linearity of the tissue digest calibration graphs near the blank level. (One paper reported The lowest background B level attained was 3.7 mg l-1 total B, which was slightly higher than that reported by other signal enhancement with both B and Be with 2% HNO3.36) A possible solution to the problem would be to utilise a constant workers.33 Vanhoe et al.33 found the background level could not be reduced below 1.7 mg l-1 B, was similar with sub-boiled HNO3 concentration throughout any analytical run, for the sample digests and blanks, calibration standards and blanks 0.14 M HNO3 and water (~2 mg l-1 B), and did not originate from the conventional sample introduction system of boro- and wash solutions, which was used coincidentally in one study.33 silicate glass.They presumed that the background B level originated from the Milli-Q water. Vanhoe et al.33 reported a substantial memory eVect with standards containing >50 mg l-1 B, lasting for ~15 min with 100 mg l-1 B. They therefore ran standard solutions at the end Calibration graphs of the run.Fig. 1 shows the result of an experiment where a set of aqueous calibration standards were run followed by three sets of Variable blanks and matrix suppression standards containing, respectively, HNO3 digests of blood, plasma and brain tissue, then a second set of aqueous stan- As mentioned previously, checks were made on background dards. The graphs were all normalised to Be and subtracted B levels during runs. A set of standard solutions was usually using the lowest aqueous blank.All the graphs were linear run first, but if the background levels fell substantially during between 5 and 80 mg l-1 B. The linear aqueous calibration a run a second calibration curve was constructed at the end graphs had regression equations y=5539x+3432 (r2=0.9996) of the run. A blank or a low standard solution (5 mg l-1 B) and y=5802x+2046 (r2=0.9990), respectively, where y is the was therefore run after every five samples. total B counts s-1 and x is the total B concentration in The use of the internal standard successfully compensated mmol l-1.for the matrix eVect produced by the high Na concentration Digest blanks contained high B concentrations either in the digests of cells solubilised in 1 M NaOH [0.05 M or because washout was not complete after the previous high 1.15 mg ml-1 Na (0.115% Na)]. Both B and Be (the internal standard or possibly due to matrix eVects. The digests con- standard) signals were suppressed by the Na, thus giving the tained 2–5% HNO3.Standard solutions were run in order of impression that the digests contained less B than the digest ascending concentration utilising a wash of 1 min with blanks. Matrix suppression of the Be signal was 69%. de-ionised water between solutions. The general wash method Similarly, the lowest dilutions (1 in 2.7) of the BPA solutions for B samples was only utilised between diVerent sample types. in 1 M NaOH analysed contained 0.37 M NaOH or Since the graphs were normalised, the non-linearity of the 8.5 mg ml-1 Na (0.85%).In this case the internal standard calibration graphs may have been caused by an increasing was Li and the Li signal was suppressed by 48%. matrix eVect at lower B concentrations, not compensated by It is well known that light analytes are more seriously the internal standard. An explanation for the non-linearity of aVected by matrix eVects than heavier analytes.45,46 However, calibration graphs near the blank level, particularly for a light suppression by 10% of the Cu, Zn, Co and Bi signals by element such as B, was suggested by Gregoire in 1990.41 The 1 mgml-1 Na was reported by Gray and Date47 and Douglas molar ratio of B to dissolved solids increases with increasing and Houk.48 Gregoire30 reported suppression of the B signal B concentration so that the matrix eVect can decrease with at >2 mgml-1 Na, with 3% suppression at 2.1 mg ml-1 Na increasing B concentration.and 12% suppression at 4.2 mg ml-1 Na. The suppression Fig. 1 also illustrates the tendency for aqueous blank levels experienced here was substantially higher, presumably because the solubilised cell digests provided a more complex Nacontaining matrix and the BPA solutions had a higher Na content. Probst et al.36 investigated the matrix eVects of single elements on B and Be count rates and found, similarly, considerable suppression with 10 mg ml-1 NaCl or NaNO3.Nyomura et al.,35 when comparing sample preparation methods, found B concentrations in biological reference materials to be slightly higher (although still within an acceptable range) in samples prepared by wet dissolution, presumed to be caused by leakage from borosilicate glass containers. They also found that B concentrations in the blanks tended to be more variable and higher when prepared by wet digestion. Evans and Kra�henbu� hl32 stressed the diYculty of obtaining accurate blank values when determining the B content of biological materials and their importance in determination by isotope dilution.For samples containing low levels of B, the digest blanks contained very similar levels of B to the sample digests. This was true for brain tissue, brain homogenate, blood and plasma digests and even after normalisation to the internal standard, blank subtraction was diYcult or impossible. This was most common with the brain tissue digests and blanks, with which Fig. 1 Boron calibration curves by ICP-MS: aqueous solutions and blood, plasma and brain tissue digests. there was matrix suppression which could not be compensated J. Anal. At. Spectrom., 1999, 14, 1545–1556 1549by the internal standard. The problem did not arise with media digests. Correction for peak overlap by carbon with the VG PlasmaQuad As mentioned previously, the VG PlasmaQuad Mark 1 only operated in the multi-channel scanning mode. For samples containing very high C levels, the 12C peak overlapped the adjacent 11B peak.For these samples it was necessary to correct for the increased counts s-1 on the 11B peak and the count rate was only measured for the part of the peak where there was no overlap, which was normally between 10.5 and 11.3 u. The same area was then measured on the B standard peaks and the mean percentage of counts s-1 for the whole peak, lying between 10.5 and 11.5 u, was calculated. Once the normal percentage of counts s-1 in this area was known, the same percentage could be added to the sample peaks where only part of the peak count rate had been measured.Use of 10B511B ratio determination for the detection and measurement of elevated 10B levels Initial sample total B and 10B concentrations were relatively high: 10B concentrations in blood 14–19 mg g-1, brain tissue 1–13 mg g-1, brain homogenate 2–249 mg g-1 and wash medium 0.007–17.2 mg ml-1. Total B concentrations were slightly higher: blood 14–19 mg g-1, brain tissue 3–19 mg g-1, brain homogenate 9–281 mg g-1 and wash medium 0.008– 18.0 mg ml-1. For these samples the use of an internal standard successfully compensated for matrix eVects.Rapid narrow mass range scans were made of sample digests for comparison. Fig. 2 illustr typical results at the beginning of the study when samples contained very high levels of 10B, whereas Fig. 3 illustrates results later in the study where elevated 10B levels were reduced.Fig. 2 shows an increasing 10B content of tissue digests (brain homogenate) following Fig. 2 10B511B ratios in brain homogenate digests by ICP-MS. initial trial incubation with 10B-enriched boric acid. The 10B concentrations in the original brain homogenate reached a maximum of 249 mg g-1. Fig. 3 illustrates higher C levels in The optimisation of the scan parameters and scan times for 10B511B ratio determination enabled instrumental DLs (using brain tissue digests than for the digests in Fig. 2; a digest blank is included for comparison. All three tissue digests in three times the standard deviation of replicate determinations made at or near the blank level ) for total B of <0.1 mmol l-1 Fig. 3 contained elevated levels of 10B, with concentrations in tissue of 1.2–13 mg g-1 after incubation with BPA. Carbon (<1 mg l-1) and for 10B of <0.01 mmol l-1 (<0.1 mg l-1) to be attained, despite the relatively high background con- levels were not high enough to prevent analysis.The high cost of the 10B-containing compounds was an centrations. The calculated sample DLs for B (10B, 11B and total B) in biological samples were as low as 0.001 mmol g-1 incentive to work at relatively low concentrations, i.e., nanomolar levels, where problems of contamination and high B (0.01 mg g-1) (10-9 M BPA solutions). The DLs attained were related to the amount of sample digest blanks and background concentrations became signifi- cant.For the analysis of samples containing low B levels, the utilised; therefore a lower DL was attained for solubilised cells of 0.0001 mmol ml-1 (0.001 mg ml-1) 10B and for the reagent digest blanks contained very similar B levels to those of the sample digests, where, even after normalisation to the internal solutions analysed 10B concentrations as low as 0.0003 mmol g-1 (0.003 mg g-1) were reported. These sample DLs standard, blank subtraction was diYcult or impossible.This was most common with brain tissue digests but was also found compare favourably with those of other workers.33,36 10B511B ratios were monitored from the beginning of the with brain homogenate, blood and plasma digests. For some samples, the results of analysis were therefore reported without study and it became clear that increases in sample 10B level could be detected using the 10B511B ratio, below the calculated digest blank subtraction or with both ‘running blank’ and digest blank subtraction.The ‘running blank’ was the instru- DL for total B or 10B. 10B511B ratio determination was suYciently precise to enable statistically significant increases mental blank with an aqueous standard blank solution. The power of stable isotope ratio determinations in detecting in 10B concentration to be detected and confirmed at these low B levels. Despite achieving low DLs, elevated 10B levels increases in 10B in situations where no total B concentrations could be accurately determined was illustrated using these could not be confirmed without the accurate determination of 10B511B ratios.Running 10B511B ratios for standard and samples for which normal blank subtraction could not be used. The mean RSD for five replicate determinations of 10B511B sample solutions were utilised for novel methods of calculating elevated 10B levels in the samples. ratio at the blank level was ~2% (range 0.5–5.2%, n=7, three diVerent analytical runs) and similar precision was obtained Signals for the two B isotopes were high enough to permit the determination of 10B511B ratios in all samples, including with low B sample digests.The precision for isotope ratio determination with the aqueous standard solutions was gener- digest blanks. The lowest background B level was ~4 mg l-1 total B, equivalent to a minimum sensitivity for total B of ally <1.5% (range 0.5–2.8%, n=10, three diVerent analytical runs).~2500 counts s-1. 1550 J. Anal. At. Spectrom., 1999, 14, 1545–1556using the mean total B counts s-1, normalised to the internal standard, obtained for the running blank (aqueous blank) and the accurate running 10B511B ratio obtained using the B standards. The lowest running blank total B concentration obtained for any run was 3.7 mg l-1. For this particular run, the running 10B511B ratio using the boron standards was 0.236 (s 0.003; RSD 1.24%; n=5), whereas if the three aqueous blanks had also been used to calculate the running 10B511B ratio, it would have been 0.242 (s 0.009; RSD 3.65%; n=8).During the same run, the running 10B511B ratio for the digest blanks was 0.248 (s 0.006; RSD 2.58%; n=4). The 10B511B ratio using the normal abundance of the two isotopes is 0.2466. Since the bias of the mass spectrometers varied slightly from day to day, a correction factor was used to normalise the running 10B511B ratio sample results, based on the running 10B511B ratio for the B standards.The correction factor assumed that, if no mass bias existed, the standards would have given a 10B511B ratio of 0.2466, since no reference materials of known 10B511B ratio were utilised. The corrected running 10B511B ratio sample results were then reported, allowing comparison between diVerent runs. For digest blank subtraction, the average intensities for the individual 10B and 11B isotopes were utilised in the normal manner. (2) Calculation of elevated 10B levels.Elevated 10B levels were calculated using the running blank subtracted results in micromoles (usually mmol g-1). It was assumed that any 10B present due to contamination or contributions from any source other than a 10B-containing compound would be related to the 11B concentration by the normal abundance of the two isotopes. This ‘blank’ level of 10B could therefore be calculated by multiplying the 11B concentration in the sample by the normal 10B511B ratio of 0.2466.The background 10B level could then be subtracted from the running blank subtracted 10B concentration to give the elevated 10B level in the sample. In this instance, using the running 10B511B ratio for the Fig. 3 10B511B ratios in tissue digests by ICP-MS. standards instead of 0.2466 gave a more accurate result. In summary, the elevated 10B concentration was given by the Calculations following equation: For most analyses by ICP-MS, a convention has developed El[10B]=S[10B]-(S[11B]×RS10B511B) that analysis is carried out with amounts measured in microwhere El[10B]=elevated 10B concentration in sample grams since the detector is more sensitive to elements with (mmol g-1), S[10B]=running blank subtracted 10B con- higher m/z values.At the high end of the m/z range, the centration in sample (mmol g-1), S[11B]=running blank relative diVerences in m/z between isotopes is small, but at the subtracted 11B concentration in sample (mmol g-1) and low end the diVerence is significant.It was therefore convenient RS10B511B =running 10B511B ratio for the standard boron to change to working in micromoles when calculating concensolutions. trations of 10B and 11B, especially at concentrations near to Occasionally it was necessary or more convenient to calcu- the DL. late the elevated 10B concentration using the normalised A new method of calculating elevated 10B levels was develcounts s-1 for B, e.g., when the sample counts s-1 for 11B oped using the determined 10B511B ratios.The method was of were lower than those obtained for the running blank. In these particular value for analysis at the DL, when blanks and cases the elevated counts s-1 for 10B were calculated in a samples contained similar B concentrations and digest blank similar manner and the elevated 10B concentration obtained subtraction could not be utilised. by dividing the elevated counts s-1 for 10B by the X coeYcient for B in counts s-1 per mmol B, using the following equation: (1) Calculation of running 10B511B ratio and running blank 10B and 11B concentrations. The ‘running 10B511B ratio’ was El[10B]= 10Bcs-1-(11Bcs-1×RS10B511B) XB the 10B511B ratio of the standards and samples which did not contain elevated levels of 10B measured during an individual where El[10B]=elevated 10B concentration in sample run.An accurate value of the running 10B511B ratio was (mmol l-1), 10Bcs-1=normalised counts s-1 for 10B (non- required for three reasons: first, for comparison with sample blank subtracted), 11Bcs-1=normalised counts s-1 for 11B 10B511B ratios in order to detect elevated levels of 10B; second, (non-blank subtracted) and XB=X coeYcient for boron to determine the elevated 10B levels in samples; and third, to (counts s-1 per mmol B).determine accurately the 10B and 11B concentrations (count rates) in the running blank. Mass bias interference The most accurate value of the running 10B511B ratio was obtained using the mean 10B511B ratio for the B standards.Assuming that the B standards utilised contained the normal abundance of the two isotopes, the mass discrimination of the The running blank 10B and 11B count rates were then calculated J. Anal. At. Spectrom., 1999, 14, 1545–1556 1551instruments was calculated using the running 10B511B ratio ised running 10B511B ratios were reported (graph j). A more detailed analysis of the data obtained with the final dilutions standard results.For the PE SCIEX Elan 5000, the initial mass bias obtained for 10B511B ratios was -11.3% (range of ~2 mg l-1 10B is given in Fig. 4(B). The eVect of B contamination from NaOH and water was -5.5 to -14.9%, n=6). After optimisation of the mass calibration, the mass bias was reduced to -6.4% (range -1.5 to lower the 10B511B ratio as shown in all three graphs. The lowest ratios were seen when the final B concentrations were to -11.4%, n=10).The mass bias appeared to be generally lower for the VG PlasmaQuad Mark 1 at -5.1% (range -2.3 2 mg l-1 (2×10-7 M). When the final (analysed) B concentrations were adjusted to 10 mg l-1, the 10B511B ratio increased to -7.0%, n=3), where scan conditions were not optimised for isotope ratio determination. Gregoire30 also obtained the as the original concentration of BPA increased, which reflected decreased contamination from NaOH as the dilution for mass bias towards the 11B isotope.Evans and Kra�henbu� hl32 obtained a greater mass bias of 20% with a PE SCIEX Elan analysis increased (graph ii). The solutions containing the highest B concentrations gave ratios lower than the true ratios 5000 when determining 11B510B ratios in biological reference materials. (graph i); for the 10-3M BPA solution diluted to 450 mg l-1 B, this reflected the saturation of the detector with 10B. Gregoire30 found matrix induced mass discrimination with 3 mgml-1 Na.There was only evidence for matrix induced The data obtained with the final dilutions of ~2 mg l-1 10B showed the power of stable isotope measurements by ICP-MS mass discrimination during this study with the lowest dilutions of solutions of boronophenylalanine in NaOH (1 in 2.7), [Fig. 4(B)]. The increase in the normalised 10B511B ratio from the baseline value of 0.247 at 1×10-9 M to 1×10-10M BPA containing 0.37 M NaOH or 8.5 mg ml-1 Na; the mass bias for 10B511B ratios was lower than -2%.(original concentration) to 0.286 at 1×10-8 Mwas statistically significant ( p<0.001). The standard solutions of 0.4–4.0 mg l-1 total B (4×10-8–4×10-7M) gave a mean RSD for five 10B511B isotope ratio determination of boronophenylalanine solutions replicate determinations of 10B511B ratio of 2.1% (range 1.9–2.5%). The precision was similar for the samples. The Solutions of BPA in 1 M NaOH from 10-3 to 10-10 M were data therefore demonstrated, despite high blanks, the possibilfurther diluted for analysis by ICP-MS in order to determine ity of working with concentrations as low as 10-8M BPA, to whether work at 10-9 MBPA was feasible.Fig. 4(A) illustrates study the uptake of 10B in tissues and cells. the resulting 10B511B ratio data. Initially the 10-3 and 10-4 M solutions were diluted for analysis to give 450 (4.5×10-5 M) 10B511B ratio determination in tissue grown with boronoand 50 mg l-1 B, respectively (graph i). The original solutions phenylalanine. A series of tissue digests were analysed.One were then diluted to give~2 mg l-1 10B and re-analysed (graph investigation included tissue samples (mass range 33–69 mg) j); HNO3 was added to neutralise the NaOH with the lowest which had been grown with increasing concentrations of BPA dilutions (10-7 to 10-10 M) as described previously. The from 1×10-8–1×10-3 M. Results for B content and 10B511B original 10-3–10-6M solutions were also diluted to ratio were not digest blank subtracted since the digest blanks ~10 mg l-1 10B for further analysis (graph ii).contained relatively high B levels (Table 3). The 10B511B ratios for the solutions containing 10 mg l-1 The results again illustrated the power of stable isotope were blank subtracted (graphs i and ii), but this was not ratio measurements by ICP-MS. The digest blanks and digests possible for those diluted to ~2 mg l-1 10B where the normal- from tissue grown with 10-5–10-8 M BPA gave normalised running 10B511B ratios at the baseline value of 0.247, but the increase to 0.300 with the tissue grown with 10-4 M BPA was statistically significant.A further increase to a ratio of 1.09 occurred with the tissue grown with 10-3 M BPA. In this particular case the 10B511B ratios calculated using the nonblank subtracted B concentrations for the two isotopes show the same trends. Elevated 10B levels could not have been detected without the accurate determination of 10B511B ratios.Fig. 5 shows narrow mass range scans of some of the tissue digests from the same investigation: the tissue digests grown with 10-3 and 10-4 M BPA (tissue digests 3 and 4) with increased 10B levels and the tissue digests grown with 10-6 and 10-8 M BPA (tissue digests 6 and 8) with normal abundance of the two isotopes. Confounding factors in 10B511B determination Confounding factors all worked against a false detection of an increased 10B511B ratio, with the exception of overloading the detector by running a sample with a very high B concentration, e.g., 450 mg l-1 B, as described previously.The confounding factors included 12C overlap on the 11B peak with sample digests containing high C levels, and matrix suppression. The majority of the medium digests in Table 6 had a slightly lower 10B511B ratio than the blanks due to 12C overlap on the 11B peak. Matrix mass discrimination also tended to decrease the 10B511B ratio. 10B content of boron-containing compounds The 10B content was determined for three diVerent boroncontaining compounds: boric acid, p-boronophenylalanine and Fig. 4 ICP-MS analysis of 10B511B ratios in boronophenylalanine solutions. carboranylacetic acid. 1552 J. Anal. At. Spectrom., 1999, 14, 1545–1556Table 3 10B511B ratio determination in tissue digests by ICP-MS: tissue grown with boronophenylalanine. The blanks used for acid digestion contained relatively high levels of boron.Results were therefore not digest blank subtracted Corrected 10B/ 11B/ Total B/ 10B/ 11B/ Total B/ Final running Sample Sample mg g-1 mg g-1 mg g-1 mmol g-1 mmol g-1 mmol g-1 10B 511B ratioa 10B511B ratio grown with Tissue 8 0.7 3.1 3.8 0.07 0.29 0.35 0.231 0.245 10-8 M BPA Tissue 7 0.4 2.3 2.7 0.04 0.21 0.25 0.198 0.251 10-7 M BPA Tissue 6 0.4 2.0 2.4 0.04 0.18 0.22 0.231 0.245 10-6 M BPA Tissue 5 0.3 1.5 1.8 0.03 0.13 0.16 0.234 0.247 10-5 M BPA Tissue 4 0.5 1.6 2.0 0.05 0.14 0.19 0.323 0.300 10-4 M BPA Tissue 3 1.4 1.0 2.4 0.14 0.09 0.23 1.57 1.09 10-3 M BPA Equivalent values for digest blanks 0.6 2.7 3.2 0.06 0.24 0.30 0.233 0.246 s 0.2 1.0 1.2 0.02 0.09 0.11 0.003 0.002 Running 10B511B ratio for the standard solutionsb 0.2331 s 0.0009 RSD (%) 0.39 aCalculated using boron calculations before rounding up.bThe running 10B511B ratio for the standard solutions indicates the normal abundance of the two boron isotopes as determined by the mass spectrometer during this run.were 1.5 mg l-1 and 2.7 mg l-1, respectively. The estimated level of 10B present in the boric acid was therefore 85%. The data in Fig. 4(A) (specifically graph i, aesults from the 1×10-4 M BPA solution diluted to 50 mg l-1 for analysis) showed that 96% of the B in the BPA sample was 10B. A sample of ~4 mg of carboranylacetic acid (CAA) was dissolved in 1 M NaOH, giving a 1.0 mg ml-1 solution, which was diluted for analysis to give 20 mg l-1 (0.099 M) and 40 mg l-1 (0.198 M) CAA.Since one molecule of CAA contains 10 B atoms, the predicted total B concentrations in the two solutions were 0.99 and 1.98 M, respectively. Analysis by ICP-MS gave total B concentrations slightly higher, 1.23 and 2.37 M. The carboranylacetic acid contained boron with a normal isotopic distribution; the normalised running 10B511B ratio obtained for the CAA solutions was 0.2450±0.0009 (mean±s). The mean running 10B511B ratio for the B standards was 0.2224±0.0009 (mean±s).Analytical precision from duplicates Analytical data for the precision from duplicates were obtained for 12 samples of blood during two runs (runs A and B), six samples of cells solubilised in 1 M NaOH and six samples of medium both analysed in the same run (run C). Table 4 gives the data from runs A, B and C using the conventional methods for calculating 10B and total B concentrations and Tables 5 and 6 give the results from the same analytical runs with the developed calculation methods utilising 10B511B ratio correction.Running blank subtracted results were reported for the blood samples in both cases since the blood digest blanks contained relatively high B levels. Only the blood samples in run B contained significant elevated levels of 10B. The 10B concentration range in the samples was <0.0002 mmol ml-1 in the solubilised cells, <0.007 mmol ml-1 in the media, <0.001 mmol g-1 in the blood samples of run A and 0.007–0.014 mmol g-1 in the blood samples of run B.The equivalent total B concentrations in the samples were Fig. 5 10B511B ratios in tissue digests by ICP-MS: uptake of boron- 0.001 mmol ml-1, <0.04 mmol ml-1, <0.002 mmol g-1 and ophenylalanine. 0.011–0.019 mmol g-1, respectively. Table 4 shows, using the conventional methods for calculation of 10B concentration, that the range and mean of within- The 10B content of a sample of boric acid was determined by measuring the elevated 10B level.The solutions analysed run RSDs for blood (only run B), solubilised cells and medium were very similar, all <17% and with means of ~7%. The contained relatively low levels of boric acid: 10 and 20 mg l-1 (0.162 and 0.323 mmol l-1), equivalent to ~1.7 and mean (range) RSDs were for blood 6.9% (1.2–16.8%), for solubilised cells 7.3% (1.5–16.6%) and for medium 6.9% ~3.4 mg l-1 B, respectively. The normalised running 10B511B ratios obtained for the two solutions were 0.496 and 0.752 (0.5–13.9%).Similarly, using the conventional methods for calculation, and the elevated 10B concentrations found in the two solutions J. Anal. At. Spectrom., 1999, 14, 1545–1556 1553Table 4 Analytical precision from duplicates for total B and 10B concentrations in blood, solubilised cells and medium Total B concentration/mmol g-1 10B concentration/mmol g-1 Type of Mean sample Sample No. 1st assay 2nd assay Mean s RSD (%) 1st assay 2nd assay Mean s RSD (%) reported Blood Run Aa,b— 1l <0.002 <0.002 <0.002 0.003 <0.001 <0.001 <0.001 0.001 2l <0.002 <0.002 <0.002 0.004 <0.001 <0.001 <0.001 0.001 Run Ba— 1W 0.019 0.019 0.019 0.0005 2.7 0.0143 0.0135 0.0139 0.0005 3.7 2W 0.019 0.014 0.016 0.003 19.9 0.0135 0.0106 0.0120 0.0020 16.8 3W 0.016 0.014 0.015 0.001 9.6 0.0099 0.0093 0.0096 0.0004 4.1 4W 0.019 0.017 0.018 0.001 4.9 0.0128 0.0125 0.0127 0.0002 1.2 5W 0.013 0.017 0.015 0.003 19.7 0.0082 0.0104 0.0093 0.0015 6.5 6W 0.014 0.016 0.015 0.002 10.4 0.0088 0.0098 0.0093 0.0007 7.3 1F 0.012 0.013 0.013 0.001 8.9 0.0083 0.0095 0.0089 0.0009 9.9 2F 0.014 0.014 0.014 0.0002 1.2 0.0092 0.0096 0.0094 0.0003 3.5 3F 0.012 0.011 0.012 0.001 10.5 0.0072 0.0068 0.0070 0.0003 4.2 6F 0.011 0.011 0.011 0.0001 0.8 0.0066 0.0068 0.0067 0.0001 1.9 Mean 8.8 6.9 Run Cc— Cells solubilised in 1 M NaOH and medium Cell digests 1 0.0011 0.0011 0.0011 0.00002 2.2 0.00018 0.00019 0.00019 0.000003 1.5 0.0002 2 0.0006 0.0006 0.0006 0.00002 3.0 0.00010 0.00011 0.00010 0.000007 7.0 0.0001 3 0.0007 0.0006 0.0007 0.00007 10.6 0.00013 0.00011 0.00012 0.000019 16.6 0.0001 4 0.0005 0.0005 0.0005 0.00002 3.5 0.00008 0.00008 0.00008 0.000006 7.2 0.0001 5 0.0005 0.0005 0.0005 0.000005 1.1 0.00009 0.00008 0.00009 0.000006 6.6 0.0001 6 0.0006 0.0006 0.0006 0 0.0 0.00010 0.00010 0.00010 0.000003 5.2 0.0001 Mean 3.4 7.3 Medium digests A 0.0261 0.0284 0.0273 0.0016 5.9 0.0044 0.0048 0.0046 0.0003 6 B 0.0238 0.0291 0.0264 0.0038 14.4 0.0041 0.0050 0.0045 0.0006 13.9 C 0.0334 0.0337 0.035 0.0002 0.6 0.0057 0.0058 0.0058 0.00003 0.5 D 0.0357 0.0389 0.0373 0.0023 6.2 0.0061 0.0067 0.0064 0.0004 6.1 E 0.0018 0.0019 0.0019 0.00005 2.6 0.0002 0.0002 0.0002 0.000008 5 F 0.0024 0.0022 0.0023 0.0001 4.3 0.0003 0.0002 0.0003 0.00003 10 Mean 5.7 6.9 aSample masses were 200–600 mg.Detection limits were 0.002–0.005 mmol g-1 for total B and 0.0002–0.0005 mmol g-1 for 10B.bVariation in digestion blank levels of boron increased detection limits for 10B in this run. cSample volumes were 1 ml. Detection limits were <0.001 mmol ml-1 for total B and 0.0001 mmol ml-1 for 10B. 1554 J. Anal. At. Spectrom., 1999, 14, 1545–1556Table 5 Analytical precision from duplicates for 10B and elevated 10B concentrations in blood. Running blank subtracted results with 10B511B ratio concentration 10B concentration/mmol g-1 Elevated 10B concentration/mg g-1 Sample No. 1st assay 2nd assay Mean s RSD (%) 1st assay 2nd assay Mean s RSD (%) Run A— 1l 0.0012 0.0009 .0010 0.0002 18.4 2l 0.0011 0.0011 0.0011 0.00002 2.2 Equivalent value for digest blanksa 0.004 0.002 Run B— 1W 0.0145 0.0138 0.0141 0.0005 3.5 0.130 0.123 0.126 0.005 4.0 2W 0.0137 0.0108 0.0123 0.0020 16.6 0.122 0.098 0.110 0.017 15.6 3W 0.0102 0.0096 0.0099 0.0004 4.3 0.085 0.083 0.084 0.001 2.0 4W 0.0130 0.0128 0.0129 0.0002 1.5 0.113 0.114 0.113 0.00001 0.01 5W 0.0085 0.0106 0.0096 0.0015 15.7 0.071 0.088 0.080 0.012 14.8 6W 0.0090 0.0100 0.0095 0.0007 7.5 0.077 0.083 0.080 0.005 6.1 1F 0.0088 0.0099 0.0094 0.0008 8.2 0.074 0.086 0.080 0.008 9.6 2F 0.0098 0.0101 0.0099 0.0002 1.9 0.081 0.086 0.083 0.003 3.7 3F 0.0076 0.0072 0.0074 0.0003 3.5 0.059 0.058 0.059 0.0007 1.1 6F 0.0069 0.0072 0.0070 0.0002 2.5 0.055 0.056 0.056 0.001 2.7 Equivalent value 0.008 0.003 for digest blanksa aRough estimate only.Table 6 Analytical precision from duplicates for running 10B:11B ratios in blood, solubilised cells and medium Corrected running 10B511B ratio Type of Sample sample No. 1st assay 2nd assay Mean s RSD (%) Blood Run A— 1l 0.257 0.233 0.245 0.017 7.0 2l 0.240 0.247 0.243 0.005 1.9 Equivalent value for digest blanks 0.259 0.007 2.6 Run B— 1W 1.84 1.66 1.75 0.13 7.2 2W 1.72 1.80 1.76 0.06 3.2 3W 1.19 1.40 1.30 0.15 11.6 4W 1.48 1.76 1.62 0.20 12.3 5W 1.16 1.23 1.19 0.05 4.2 6W 1.29 1.17 1.23 0.08 6.7 1F 0.94 1.22 1.08 0.19 17.9 2F 0.88 1.12 1.00 0.17 17.3 3F 0.84 0.87 0.86 0.02 1.8 6F 0.88 0.84 0.86 0.03 3.1 Equivalent value for digest blanks 0.269 0.004 1.4 Solubilised cellsa Run C— 1 0.264 0.263 0.263 0.001 0.4 2 0.265 0.275 0.270 0.007 2.7 3 0.274 0.267 0.270 0.005 0.7 4 0.261 0.264 0.263 0.002 1.8 5 0.275 0.269 0.272 0.005 0.9 6 0.266 0.269 0.268 0.002 0.9 Equivalent value for digest blanks 0.276 0.006 2.3 Medium Run C— A 0.233 0.233 0.233 0.002 0.05 B 0.236 0.234 0.235 0.001 0.5 C 0.236 0.236 0.236 0.0003 0.1 D 0.237 0.237 0.237 0.0001 0.06 E 0.186 0.176 0.181 0.007 3.8 F 0.190 0.180 0.185 0.007 3.8 Equivalent value for digest blanks 0.287 0.002 0.7 aCells were solubilised in 1 M NaOH.J. Anal. At. Spectrom., 1999, 14, 1545–1556 155511 Y. Mishima, M. Ichihashi, M. Tsuji, S. Hatta, M. Ueda, the range and mean of within-run RSDs for total B in blood, C. Honda and T. Suzuki, J. Invest. Dermatol., 1989, 92, 321S. solubilised cells and medium were more variable but all were 12 J.A. Coderre, J. Kalef-Ezra, R. G. Fairchild, P. L. Micca, L. E. <20% with means of 3–9% (Table 4). This was probably a Reinstein and J. D. Glass, Cancer Res., 1988, 48, 6313. result of the variable presence of residual carbon in the digested 13 J. A. Coderre, J. D. Glass, S. Packer, P. L. Micca and samples giving rise to overlap of the 12C peak on the adjacent D. Greenberg, Pigm. Cell Res., 1990, 3, 310. 14 S. Packer, J. A. Coderre, S.Saraf, R. Fairchild, J. Hansrote and 11B peak. For total B, the mean (range) RSDs were for blood H. Perry, Invest. Ophthalmol. Vis. Sci., 1992, 33, 137. 8.8% (1.2–19.9%), for solubilised cells 3.4% (0–10.6%) and 15 K. Z. Matalka, M. Q. Bailey, R. F. Barth, A. E. Staubus, D. M. for medium 5.7% (0.6–14.4%). Adams, A. H. Soloway, S. M. James and J. H. Goodman, in Specific results could not be reported for the blood samples Progress in Neutron Capture Therapy for Cancer, eds.B. Allen, D. in run A using the conventional methods for calculations since Moore and B. Harrington, Plenum Press, New York, 1992, p. 429. the B concentrations were lower than the calculated DLs, but 16 B. J. Allen, J. K. Brown, M. H. Mountford, S. R. Tamat, A. Patwardan, D. E. Moore, M. Ichihashi, Y. Mishima and could be reported using the developed calculation methods S. B. Kahl, Strahlenther. Onkol., 1989, 165, 163. utilising 10B511B ratio correction (Table 5). Table 5 gives the 17 O.Leukhart, M. Caviezel, A. Eberle, E. Escher and A. Tun-khi, running blank subtracted results with 10B5 11B ratio correction Helv. Chim. Acta, 1976, 59, 2184. for the duplicate blood samples and the equivalent concen- 18 M. F. Hawthorne, R. J. Wiersema and M. Takasugi, J. Med. trations for the digest blanks, with the calculated elevated 10B Chem., 1972, 15, 449. concentrations in the blood samples of run B. There was only 19 R. F. Barth, A. H. Soloway, R. G. Fairchild and R.M. Brugger, Cancer, 1992, 70, 2995. a slight diVerence between the recalculated 10B concentrations 20 G. Ko� hler and C. Milstein, Nature (London), 1975, 256, 495. and the original results and the mean (range) precision for 21 R. G. Fairchild, D. Gabel, B. Laster, D. Greenberg, W. Kiszenick duplicate analyses was slightly improved, 6.5% (1.5–16.6%). and P. L. Micca, Med. Phys., 1986, 13, 50. The mean (range) precision (RSD) for the elevated 10B levels 22 W. B.Clarke, M. Koekebakker, R. D. Barr, R. G. Downing and (mg g-1 10B) in the blood samples was 6.0% (0.01–15.6%). R. F. Fleming, Appl. Radiat. Isotop., 1987, 38, 735; Int. J. Radiat. The most useful information obtained using the developed Appl. Instrum., Part A. 23 D. E. Moore, J. Pharm. Biomed. Anal., 1990, 8, 547. calculation methods for identifying increased 10B level was the 24 K. Yoshino, M. Okamoto, H. Kakihana, T. Nakanishi, running 10B511B ratio and the elevated 10B level.Comparisons M. Ichihashi and Y. Mishima, Anal. Chem., 1984, 56, 839. of RSDs for running 10B511B ratios for duplicate samples with 25 F. Salinas, A. M. Pena, J. A. Murillo and J. C. J. Sanchez, Analyst, relevant data are made in Table 6. For the running 10B511B 1987, 112, 913. ratio, the precision was better for the solubilised cells and 26 C. J. Cook, S. V. Dubiel and W. A. Hareland, Anal. Chem., 1985, medium, which showed a normal abundance. The mean (range) 57, 337. 27 W.B. Clarke, C. E. Webber, M. Koekebakker and R. D. Barr, RSDs for duplicates samples were for blood 6.5% (1.8–17.9%), J. Lab. Clin. Med., 1987, 109, 155. for solubilised cells 1.2% (0.4–1.8%) and for medium 1.4% 28 S. R. Tamat, D. E. Moore and B. J. Allen, Anal. Chem., 1987, (0.05–3.8%). The ratios for the medium digests were slightly 59, 2161. lower than those for the digest blanks and standards, owing 29 W. F. Bauer, D. A. Johnson, S. M. Steele, K. Messick, D. L. to the high C levels in the digests, leading to a slight overlap Miller and W. A. Propp, Strahlenther. Onkol., 1989, 165, 176. of the 12C peak on the 11B peak. The data for elevated 10B 30 D. C. Gregoire, Anal. Chem., 1987, 59, 2479. 31 F. G. Smith, D. R. Wiederin, R. S. Houk, C. B. Egan and R. E. concentrations in the blood samples from run B are given in Serfass, Anal. Chim. Acta, 1991, 248, 229. Table 5, since they were the only duplicate samples with 32 S. Evans and U. Kra�henbu� hl, J. Anal. At. Spectrom., 1994, 9, elevated 10B levels, where the RSDs for duplicate samples were 1249. 5.6% (0.01–15.6%). 33 H. Vanhoe, R. Dams, C. Vandecasteele and J. Versieck, Anal. Chim. Acta, 1993, 281, 401. We thank Caroline Dore� for assistance with statistical analysis. 34 S. Evans and U. Kra�henbu� hl, Fresenius’ J. Anal. Chem., 1994, 349, 454. 35 A. M. S. Nyomura, R. N. Sah, P. H. Brown and R. O. Miller, Fresenius’ J. Anal. Chem., 1997, 357, 1185. References 36 T. U. Probst, N. G. Berryman, P. Lemmen, L. Weissfloch, T. Auberger, D. Gabel, J. Carlsson and B. Larsson, J. Anal. At. 1 Handbook of Inductively Coupled Plasma Mass Spectrometry, ed. Spectrom., 1997, 12, 1115. K. E. Jarvis, A. L. Gray and R. S. Houk, Blackie, Glasgow, 37 H. P. Schwartz, E. K. Agyel and C. C. McMullen, Earth Planet. 1992, p. 341. Sci. Lett., 1969, 6, 1. 2 D. N. Slatkin, Brain, 1991, 114, 1609. 38 M. Shima, J. Geophys. Res., 1963, 67, 911. 3 A. Mill, New Sci., 18 Nov., 1989, No. 1691, 56. 39 Handbook of Geochemistry, ed. K. H. Wedepohl, Springer Verlag, 4 M. Javid, G. L. Brownell and W. H. Sweet, J. Clin. Invest., 1952, New York, 1969. 31, 603. 40 A. J. Spivack and J. M. Edmond, Anal. Chem., 1986, 58, 31. 5 H. B. Locksley and L. E. Farr, J. Pharmacol. Exp. Ther., 1955, 41 D. C. Gregoire, J. Anal. At. Spectrom., 1990, 5, 623. 114, 484. 42 R. S. Houk, Anal. Chem., 1986, 58, 97A. 6 W. H. Sweet, A. H. Soloway and G. L. Brownell, Acta Radiol., 43 I. L. Shuttler and H. T. Delves, Analyst, 1986, 111, 651. 1963, 1, 114. 44 F. Vanhaecke, H. Vanhoe, C. Vandecasteele and R. Dams, Anal. 7 A. H. Soloway, H. Hatanaka and M. A. Davis, J. Med. Chem., Chim. Acta, 1991, 244, 115. 1967, 10, 714. 45 D. C. Gregoire, Appl. Spectrosc., 1987, 41, 897. 8 R. L. Jr. Sneath, J. E. Wright, A. H. Soloway, S. M. O’Keefe, 46 D. C. Gregoire, Spectrochim. Acta, Part B, 1987, 42, 895. A. S. Dey and W. D. Smolnycki, J. Med. Chem., 1976, 19, 1200. 47 A. L. Gray and A. R. Date, Analyst, 1983, 108, 1033. 9 M. Tsuji, M. Ichihashi and Y. Ishima, Jpn. J. Dermatol., 1983, 48 D. J. Douglas and R. S. Houk, Prog. Anal. At. Spectrosc., 1985, 93, 773. 8, 1. 10 Y. Mishima, C. Honda, M. Ichihashi, H. Obara, J. Hiratsuka, H. Fukada, H. Karashima, T. Kobayashi, K. Kanda and K. Yoshino, Lancet, 1989, 2, 388. Paper 9/03097B 1556 J. Anal. At. Spectrom., 1999, 14, 154