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Anal. Chem. 1990,62,2636-2639
(16) Allonen. H.; Zlegler, G.; Klotz, U. Clin. h r m a c o l . Ther. 1981, 30, 653-66 1. (17) Greenbiatt, D. J.; Locniskar, A.; Ochs, H. R.: Lauven, P. M. AnesthesiOlogy 1981, 5 5 , 176-179. (18) Puglisi, C. V.; Meyer, J. C.; Darconte, L.; Brooks, M. A.; DeSika, J. A. F. J. Chmmatogr. 1978, 745, 81-96. (19) Cox, R . A.; Crlfasi, J. A.; Dickey, R . E.; Ketzler, S.C.; Pshak, G. L. J. Anal. Toxicol. 1989, 13,224-228. (20) Vaslliades, J.; Sahawneh, T. H. J. Chromatogr. 1982, 228, 195-203. (21) Vaslllades. J.: Owens, C. J. Chromatogr. 1980, 782, 439-444. (22) Ferslew, K. E.; Hagardorn, A. N.; McCormick, W. F. J. Forensic Sci. 1989. 34,249-257. (23) DOUet-coassolo. c.; Aubert, C.; Coassob. P.; Cano, J. P.J. Chromatour. 1989. 487. 295-311. - (24) J s e s , C:E., Jr.; Wians, F. H., Jr.; Martinez, L. A.; Merritt, G. J. Clin. Chem. 1989. 35. 1394-1398. (25) Maurer, H.: Pfleger, K. J. Chromatogr. 1987, 422, 85-101. (26) Rubio, F.: Mlwa, E. J.: Garland, W. A. J. Chromatogr. 1982, 233, 157-165. (27) Valko. K.; Olajos. S.; Cserhati. T. J . Chromatogr. 1990, 499, 36 1-37 1. (28) Blackett, A,; Dhillon, S.;Cromrty, J. A.; Horne, R.; Richards, G. J. Chromatogr. 1988, 433,326-330. (29) Minder, E. 1.; Schaubhut, R.; Minder, C. E.; Vonderschmitt, D. J. J. Chromatogr. 1987, 479, 135-154. (30) Kaeferstein, H.; Stlcht, G. Beitr. M e d . 1988, 4 4 , 253-261. (31) Puglisi. C. V.; Pao, J.; Ferrara, F. J.; DeSilva, J. A. F. J . Chromatogr. 1985, 344, 199-209. (32) Vree. T. 8.; Baars, A. M.; Booij, L. H. D.; Driessen. J. J. Arzneim.Forsch. 1981, 37,2215-2219. (33) Vasillades, J.; Sahawneh, T. H. J. Chromatogr. 1981, 225, 266-271. (34) Altunkaya, D.; Smith, R. N. Forensic Sci. I n t . 1988. 39, 23-37. (35) Taylor, J. J. Anal. TOXiCOl. 1988, 72,53. (36) Sise, J. A,; Sharman. J. R . N. 2 . J . M e d . Lab. Techno/. 1988, 42, 46-5 1. (37) Gruhl, H. 2.Rechtsmed. 1987, 9 8 , 221-226. (38)Schuetz, H.; Schneider, W. R. 2.Rechtsmd. 1987, 99, 181-190. (39) Dixon. R.; Lucek, R.; Todd, D.; Walser, A. Res. Commun, Chem. Patho/. h r m a r O 1 . 1982, 37, 11-20. (40) Schuek H.; Borchert, A.; Holland, E. M.; Schneider, W. R.; Schoelermann, K. Be&. cierlchtl. Med. 1988, 4 6 , 149-153.
.-
(41) Wolf, S. Fresenius' 2.Anal. Chem. 1989, 333,624-628. (42) Bhattacharyya, P. K.; Grant, A. Anal. Chim. Acta 1982, 742, 249-257. (43) Orive, M. M.: Gallo, 6.; Aionso, R. M.; Vicente, F.; Vire, J. C.; Patriarche, G. J. Mlkrochim. Acta 1989, 7 , 181-190. (44) Gallo, 8.; Alonso, R. M.: Vicente, F.; Vire, J. C.; Patriarche, G. J. Anal. Lett. 1988, 27, 1211-1220. (45) Sengun, F. I.: Alper, Y.; Fedai, I.; Aksu. B. Fresenius' 2. Anal. Chem. 1985, 327,671-675. (46) Vlre, J. C.; Hermosa, B. G.; Patriarche, G. Anal. Chlm. Acta 1987, 796,206-212. (47) Vlre, J. C.; Hermosa, B. G.; Patriarche, G. J. Anal. Lett. 1988, 79, 1839- 1851. (48) Kir, S.; Onar, A. N.; Temizer, A. Anal. Chim. Acta 1990, 229, 145-147. (49) Smyth, W. F., Ed. Electroanalysis in hyglene, environmental, clinical and pharmaceutical chemistry; Analytical Chemlstry Symposia Series; Elsevier Scientific Publishing Co.: Amsterdam, 1980; Vol. 2. (50) Ribes, A.; Osteryoung, J. J . Elecboanal. Chem. 1990, 287, 125-147. (51) Hernandez, L.; Zapardiel, A.; Perez Lopez, J. A,; Bermejo, E. Taianta 1988. 35,287-292. (52) S h a h I.; Lewinson, J. Anal. Chem. 1981, 33, 187-189. (53) Wang, J. Electroanalylical Chemistry; Bard. A. J., Ed.; Marcel Dekker: New York, 1989; Vol. 16. (54) Lovric, M. J. Electroanal. Chem. 1984, 781, 35-49. (55) Lovrlc, M.; Komorsky-Lovric, S.;Murray, R. W. Electrochim. Acta 1988, 33,739-744. (56) Walser, A.; Benjamin, L. E.; Flynn, T.; Mason, C.; Schwartz, R.; Fryer, R. I. J . Org. Chem. 1978, 43,936-944. (57) Ribes, A,; Osteryoung, J. Unpublished results. (58) Webber, A.; Shah, M.; Osteryoung, J. Anal. Chim. Acta 1983, 754, 105-119. (59) Webber, A.; Shah, M.; Osteryoung, J. Anal. Chim. Acta 1984, f57, 1-16.
RECEIVED for review May 25, 1990. Accepted September 4, 1990. This work was supported in part by the National Science Foundation under Grant No. CHE8521200.
Determination of Fluoride Ion in Animal Bone by Microdiffusion A naiy sis Rikio Ikenishi,* Mamoru Kanai, Masato Ishida, Akihiro Harihara, Shinji Matsui, Isao Yahara, and Takayasu Kitagawa Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan
A mlcrodmudon analysls method for the determtnatlon of fluorlde Ion In anlmal bone sample was established by using a new dMuskn cell. The cell Is equ@pedwith a water Jacket to control the temperature. The space Inside the cell Is dlvlded by a croswbbeam stand Into top and Mom parts. I n the bottom part, derlvatlzation of fluorlde Ion to trlmethylfluoroslane (TMFS) by hexamethytdtslloxane as the reagent occurs, with the volatUe TMFS being absorbed Into alkali placed In an open-top cup supported by the stand. The cell Is placed on a magnetlc stirrer to rotate the stlrrlng bars In both reactlon and absorption solutions. Stlnlng and elevation of temperature Increase the reactlon rates. Malntalnlng the cell at 40 O C for 3 h Is sufflclent for complete extraction of fluorkle ion from the bone and Its recovery. With our method, more than 10 pg/g of fluoride Ion In bone can be m y e d with a coefflclent of varlatlon of leos than 3 % . The fluorlde levels of various rat bones were clarlfled.
We previously established a gas chromatographic method for assaying fluoride ion in animal samples, plasma, blood corpuscles, and urine (I,2). Since some clinically used fluo-
rine-containing drugs release fluoride ion as a result of metabolism in humans, it is our concern from the viewpoint of drug safety to distinguish the fluoride released from the drug from endogenous fluoride. When administered to an organism, fluoride shows strong affinity for bone and 50430% of that given by intravenous injection is absorbed. When a high dose of fluoride ion is taken for a long period, bone toxicity occurs, such as mottled teeth and osteosclerosis ( 3 , 4 ) . Our attention was attracted to determining the endogenous fluoride content in animal bone, as a reference level when determining the exogenous fluoride from a drug. The microdiffusion analysis method for measuring fluoride has generally been applied to solid samples, such as bone, ore, and various materials for dentistry. Fluoride is dissolved in a strong acid to produce HF, which is converted to trimethylfluorosilane (TMFS) with hexamethyldisiloxane (HMDS) as a derivatization reagent. Volatile TMFS, which is diffused in a reaction vessel, is trapped in aqueous alkaline solution. The reactions are (CH3)3SiOSi(CH3), 2H+ + 2F2(CH )&3iF H20
HMDS
(CH,),SiF
-
+
+ NaOH
0003-2700/90/0362-2636$02.50/00 1990 American Chemical Society
-
NaF
T ~ F S
+ (CH,),SiOH
+
(1) (2)
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
1
2
3
4
5
6cm
a Figure 1. Microdiffusion cell: (1) opening for injection of reagent; (2) polyethylene packing seal; (3) screw cap; (4) polypropylene cup for trapping solution; (5) crossed-beam stand; (6) compartment for sample; (7) polyethylene-coated stirring bar; (8) magnetic stirrer.
I
Fluoride ion, which is taken up in the alkaline solution, can be determined by using the fluoride ion selective electrode method. Diffusion accelerates greatly with the derivatization to TMFS rather than by absorption of H F gas. This principle is widely used as a microdiffusion analysis method. Many types (shapes, sizes) of diffusion vessels have been used to perform the above reactions (5-10). They commonly have two compartments in which reactions 1and 2 occur independently. Sara et al. ( 6 )fabricated a diffusion dish by using large and small acrylic boxes. A box for the alkaline solution was glued onto the inner bottom of the large box. Reactions 1 and 2 occur outside and inside of the small box, respectively. Other types of vessels, tubes, or capsules have been reported (11, 12) but methods using them require very long reaction times of from overnight to 16 h. In order to quicken the reaction rate, Sara et al. placed a diffusion dish on an agitation board which was rocked within a preset angle by a motor. Another problem has been the laborious and troublesome fabrication of the diffusion vessel. In this paper, we describe a new diffusion cell that overcomes these problems. The cell we devised has a threaded lid and a water jacket of polypropylene. A cup for trapping TMFS can be placed on a crossed-beam stand suspended by the inner wall of the cell. The cross section of the cell is shown in Figure 1. The sample is placed at the bottom of the cell, where reaction 1 occurs on addition of the reagent HMDS from a cylindrical pipe on the lid. Stirring bars are put a t the bottom of both the cell and the cup. Stirring with a magnetic stirrer quickened the reaction rates of both solutions simultaneously with elevating the temperature by circulating hot water in the jacket. Maintaining the cell a t 40 "C for 3 h is sufficient to complete the recovery of fluoride from bone at 40 "C. Another type of jacket can be attached to this diffusion cell to assay six bone samples simultaneously. The jacket has six cells and can be used with a Magnestir with six magnetic stirrers. Our method has several advantages, the first being the easy handling of the diffusion cell: the inside of the cell is hermetically sealed by screwing the lid in place of vaseline sealing or taping. Another advantage is the rapidity of the analysis, only 3 h. Also, there is no worry of contamination between the reaction and absorption solutions in the cell. We used this method to assay fluoride ion levels in various bones in the rat body. The results showed that fluoride concentration increases with age. EXPERIMENTAL SECTION Diffusion Cell. A new diffusion cell was constructed from a cylindrical polypropylene bar (Araki Rubber Co., Ltd., Japan) using a lathe (Aichi Mitsubishi Co., Ltd., type HL-300, Japan). The cell shown in Figure 1 had an internal diameter of 5 cm at
2637
the top and 3.5 cm at the bottom. This cell could be opened and shut with a threaded lid made of polypropylene. The lid was bored near its circumference and an upwardly projecting cylindrical pipe was added to introduce the reagent (HMDS solution). The pipe could be opened and shut with a small threaded lid. Polyethylene sheet packing was put on the inside surface of the two lids to prevent the gas from leaking. The outside of the cell was covered with a jacket threaded in. The jacket with two taps allowed water to enter and exit. The crossed-beam stand was suspended by setting its four edges on the shoulder of the cell. A frust-conical cup (36 mm in diameter at the opening, 26 mm in diameter at the bottom, and 30 mm high) was put on the crossed-beam stand, which was positioned to maintain a sufficient space between the bottoms of the cup and the cell. Stirring bars coated with polyethylene film were put in both the cell and the cup. The jacket with six cells was also constructed of acrylic resin. It could be put on a Magnestir, Model MGS-06 (Shibata Kagakukiki Industry Co., Ltd., Japan), with six magnetic stirrers. The jacket had six threaded openings where the cell described above could be screwed in place for six assays to be conducted simultaneously. Apparatus. A Radiometer PHM 84 research pH meter was used for potential measurement. An Orion fluoride selective electrode, type 96-09, was connected with the potentiometer. A Haake thermostat, type D1-G, circulated water at a constant temperature. Reagents and Materials. Hexamethyldisiloxane (HMDS) was purchased from Wako Pure Chemical Ind., Ltd. (reagent grade). Other chemicals were of special grade. 2.7 N HClO, Solution. Pipet 118 mL of 70% perchloric acid into a 500-mL volumetric flask set in an ice bath and dilute by adding a small portion of HzO with stirring to the mark. HMDS-HC104 Solution. Place 2 mL of HMDS into 2.7 N HClO, (ca. 35 mL) in a centrifuge tube and shake with a shaker (Iwaki KM Shaker, type V-S) for 10 min. Allow this to stand for 1 h, then aspirate the upper layer off. Total Ion Strength Adjustment Buffer Solution. Dissolve 57.8 g of NaN03, 0.3 g of trisodium citrate dihydrate, and 57 mL of acetic acid in 500 mL of HzO, and then adjust the pH to 5.3 by adding 5 N NaOH. Transfer this solution to a 1-L volumetric flask and dilute to the mark with HzO. Water used for the reagent solution was distilled from deionized water. Standard Solution of NaF. Accurately weigh about 4.5 g of NaF in a 250-mL volumetric flask and dilute it to the mark with H,O. Prepare several solutions with required concentrations by diluting the stock solution with HzO (all concentrations in this paper refer to F-). Sample Preparation. Male and female Sprague-Dawley rats (Clea Japan, Inc.) were used. Bones removed from the rats were cleaned by scraping off adhering tissues with tweezers and gauze. They were then exposed to the sun and dried until the weight became constant. The bones were then crushed into small pieces with a stainless steel mortar and pestle. Incineration of Bone Sample. The bone sample was placed on a platinum boat and put into a quartz glass tube. The tube was set in a tubular combustion furnace (Daikoh Seisakusho, Kyoto, Japan), which was heated at 500 "C for 2 h. Assay Procedure. Weigh accurately 5-100 mg of powdered bone and place it into the diffusion cell. Place a polyethylenecoated stirring bar into the cell. On the crossed-beam stand, put the gas-trapping cup in which 0.1 N NaOH solution (2 mL) and a stirring bar have been placed (measure the total weight of the cup). Shut the lid of the cell and allow water heated to 40 "C to flow into the jacket. Add 5 mL of HMDS-HC104 solution to the cell through the cylindrical pipe on the lid with a syringe. Immediately shut the small lid and stir for 3 h at 40 "C. Open the large lid of the cell and remove the cup. After the cup cooled to room temperature, weigh the cup and calculate the amount of evaporated H20from the amount of 0.1 N NaOH. Transfer 1mL of the trapping solution into an 8-mL polypropylene tube and neutralize it with 0.1 mL of 1 N HNOB. Add 1 mL of TISAB buffer solution. Immerse the fluoride-selective electrode, stir the solution with a stirrer equipped with a potentiometer, and measure the electromotive force (mV) after 5 min. (Read the mV value after at least 10 min when less than M of fluoride is used.)
2638
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
1
Table 1. Recovery of Fluoride Ion from Bone Meal of Various Sample Sizesa sample size, mg
found, ppm
6.4 26.1 61.6 76.5 104.2
1391 1311 1369 1359 1385
mean
1363
2
Relative standard deviation is 31.7. Pipet 1 mL each of the standard solutions into the polypropylene tube and successively add 1 mL of 0.2 N NaOH, 0.2 mL of 1 N HN03, and 2 mL of TISAB buffer solution. Measure the electromotive force under the same conditions. Plot the electromotive force against the logarithmic fluoride concentration to make a calibration curve. Calculate the content (gg/100 mg) in the bone sample according to the equation fluoride content (pg/lOO mg) = [found concn (M) X vol of trapping solution (mL) X 2.1 X 19.0 X 100]/[weight of bone sample (mg) x The volume of the trapping solution is calculated by subtracting the amount of evaporated H20 from 2.0 mL, the initial volume of 0.1 N NaOH.
RESULTS AND DISCUSSION Reaction Conditions in the Diffusion Cell. Effect of Temperature on Reaction Rate. Fluoride ion was derivatized to volatile TMFS of bp 16.4 "C by reaction with HMDS. The TMFS diffused into the cell and was absorbed by the 0.1 N NaOH in it for subsequent conversion to the original fluoride ion. In order to enhance the rate of the derivatization and absorption reactions, the effect of temperature was examined. Fluoride ion in each 0.05 mL of the standard solutions (1 x M as F)was reacted with HMDS and 1 X by adding the saturated HC10, solution (2.7 N) via the cylindrical pipe on the lid of the cell. After the cell had been allowed to stand for 1 h a t 25, 40, and 50 "C, the fluoride trapped in the 0.1 N NaOH was assayed. The recovery of the 1 X M sample was 81-95% at 25 "C, but rose to 100% at 40 and 50 "C. The reaction rate for the 1 X M sample was a little lower. The recovery values were about 70% at 25 "C and 8647% at 40-50 "C. However, when the reaction was carried out at over 40 "C for more than 2 h, the recovery increased to 100%. Effect of Agitation on Recovery Rate. Two solid samples, CaF, and bone sample (2.5 mg and 100 mg), were analyzed while this cell was heated to 40 "C. Figure 2 shows the percent of fluoride ion recovered over more than 0.5 h. The reactions for both samples were completed in about 2 h with stirring but were slower with less fluoride ion being obtained when stirring was not done. Constant recovery was obtained
1 C.Fa 2Boo
with stirring -c without stirring
Flgwe 2. Effect of a g i t a h on recovery of fluoride ion from CaF, and bone sample: (0)with stirring: (m) without stirring.
for bone samples with reaction times of 2-24 h, suggesting complete recovery of the F- present. When an aqueous solution of fluoride ion was allowed to react, little difference in rate was observed with and without stirring. Effect of Cup Size on Diffusion Speed. The absorption rate was greatly dependent on the surface area of the trapping solution (0.1 N NaOH) in the cup. Two cups with different diameters (18 mm and 36 mm) were tested. TMFS absorption by 0.1 N NaOH was more rapid from the wide-mouthed cup (36 mm) than from the narrow-mouthed one (18 mm). After 2 h, recovery with the former cup was complete, but was only ca. 80% with the latter cup. Effect of Reagent Concentration on the Reaction of Fluoride in Bone. HC104. Rat bone powder (100 mg) was placed into the diffusion cell and three HMDS-HC104 solutions with different acid concentrations (1,2.7, and 5 N) were added. The mixture was allowed to react a t 40 "C for 3 h. The three reagents yielded the same content of fluoride ion (153.1 f 1.9 pg/lOO mg) after the assay. NUOH.TMFS, which was produced from the bone powder by addition of HMDS-HCI04 solution (2.7 N), was absorbed by NaOH at three concentrations (0.01, 0.1, and 1 N). Recovery became constant at over 0.1 N. Less than half of the fluoride content was obtained with 0.01 N. The reagent concentrations of HC104 and NaOH employed for the assay of 100 mg of bone were 2.7 and 0.1 N, respectively. Sample Preparation. The dried bone was broken and crushed into small pieces to make one sample and then ground further to prepare another sample. No difference in the recovery of fluoride was observed between the two samples. Sample Size. Bone powder samples of various sample sizes (6-104 mg) were assayed. The assay value for all the samples was invariably constant (parts per million), as shown in Table
Table 11. Concentration of Fluoride Ion in Female Rat Bones 11 Weeks after Birth
bone
femur
left right
tibiab
left
humerus
right left
right head' lower jawc vertebrad
1
2
57.0 56.3 52.1 52.3 54.9 54.2 42.9 45.1 53.4
60.8 60.6 56.1 55.2 58.1 56.9 47.3 48.5 57.9
fluoride ion, pg/lOO mg rat no. 3 4 53.4 53.7 48.2 49.4 52.4 52.5 42.6 43.3 50.4
Relative standard deviation. Including fibula. Including tooth and fang.
52.4 52.8 49.1 48.9 53.1 52.5 40.8 41.5 48.5
5
mean f sa
43.9 43.4 39.6 39.3 42.6 42.8 33.6 35.0 40.3
53.5 f 6.3 53.4 f 6.3 49.0 f 6.1 49.0 & 6.0 52.2 f 5.8 51.8 f 5.3 41.4 f 5.0 42.7 f 5.0 50.1 f 6.5
From atlas to sixth lumbar vertebra.
Anal. Chem. 1990. 62. 2639-2643
A
m
i
o!
5
10
15
20
25
30
315
Week after b i r t h Figure 3. Change o f fluoride ion concentration in rat bone wtth age: (0)male; (0)female. I. Fluoride ion in each sample was precisely quantified irrespective of the sample size, showing that the assay data correspond to the whole fluoride present in the bone as the ion. Recovery of Fluoride Ion from Bone. The fluoride ion content in dried rat bone recovered at the specified reaction times (Figure 2) remained constant after more than 2 h at 40 "C with no increment of fluoride ion found after 24 h. This indicated that all of the fluoride in the bone could be recovered by 2 h of reaction time at 40 "C. Bone sample was ignited at 500 "C for 2 h for ashing. The fluoride in the ashes was assayed by the microdiffusion method. The assayed values were compared with those obtained by the text method without incineration. Fluoride in the incinerated femur sample of 23-week-rat (male) was determined to be 80.0 f 0.81 pg/lOO mg (n = 3). The fluoride measured by the text method was 80.9 f 1.00 pg/lOO mg (n = 3). The fluoride values obtained by the two methods showed good agreement. Thus, the fluoride was fully recovered from the bone sample by our microdiffusion analysis method. Fluoride Levels of Various Bones in Rat. Various bones were removed from 11-week-old rats and divided into the
2639
femur, tibia (including fibula), humerus, skull and the part of lower jaw in skull (including teeth and fangs), and vertebra (from atlas to sixth lumbar vertebra). The first three bones were separated into those from the right and left sides of the body. Table I1 shows that fluoride ion in the bones of five rats were present in the range of 41-54 pg/lOO mg. The amount was slightly lower in the skull. Little or no difference was observed between bones of the right and left sides. Change of Fluoride Ion Content with Rat Age. Femur bone samples were collected from rats of 3 to 33 weeks that had been bred under the same conditions. Figure 3 shows that the fluoride ion content in the femur differed greatly between the young and adult animals, increasing with age. This indicates that fluoride from food intake accumulates in the bone.
ACKNOWLEDGMENT The authors thank Mr. Yuichi Ushioda for his advice and helpful discussions. Registry No. F,16984-48-8. LITERATURE CITED (1) Ikenishi, R.; Kitagawa, T.; Nishiuchi, M.; Takehara, K.; Yamada, H.; Nishino, I.; Umeda, T.; Iwatani, K.; Nakagawa, Y.; Sawai, M.; Yamashita, T. Chem. Pharm. Bull. 1988, 36, 662-669. (2) Ikenishi, R.; Kitagawa, T. Chem. pharm. Bull. 1988, 36, 810-814. (3) Weathereli, J. A. Handbook of Experimental Pharmacology; SpringerVerlag: Berlin, 1966; Vol. 20, Part I,pp 141-172. (4) Smith, G. E. Sci. TofalEnviron. 1985, 43, 41-61. (5) Taves, D. R. Talanfa 1968, 15, 969-974. (6) Sara, R.; Wanninen, E. Talsnta 1975, 22, 1033-1036. (7) Yoshda, M.; Makihara, Y.; Katswa, T. Nihon Kagaku Kaishi 1978, 10, 1375-1379. (8) Charen, J.; Taves, D. R.; Stamm, J. W.; Parkins, F. M. Calcif. Tissue Int. 1979, 27, 95-99. (9) Hattab, F.; Frosteil, G. Acta Odontol. S a n d . 1980, 38, 385-395. (10) Kobayashi, S. Kdtoebeigaku-zasshi 1982, 32, 51-69. (11) Hailsworth, A. S.;Weathereli, J. A. Dtsch. D . Anal. Chem. 1976, 48, 1660-1664. (12) Reddy, J.; Grobler, S. R. J . Clin. Periodontol. 1988, 15, 217-221.
RECEIVED for review April 24,1990. Accepted August 23,1990.
CORRESPONDENCE Photodissociation-Photoionization Mass Spectrometry of n -0ctene Isomers Sir: One long-standing problem in analytical mass spectrometry is distinguishing isomeric structures. Often times, isomeric cations have low energy barriers for rearrangement that cause them to lose memory of the initial ion structure prior to fragmentation. One approach toward overcoming this problem is to probe neutral rather than ionic fragmentation, since the barrier for rearrangement of the neutral precursor is frequently much higher than that of its ionic counterpart (I). We propose a new method for studying neutral decomposition by mass spectrometry, photodissociation-photoionization (PDPI). Neutral molecules are photodissociated in the source region of a time-of-flight mass spectrometer with a pulsed excimer laser beam. After an appropriate time delay, a vacuum ultraviolet beam traverses the same region and softly ionizes the dissociation products and any remaining undissociated parent molecules. The overall process is summarized in Scheme I. As in conventional mass spectrometry, the mass 0003-2700/90/0362-2639$02.50/0
Scheme I
M
u2
>M
+
spectrum contains both the parent ion and various fragment ions. However, unlike conventional mass spectrometry, fragment ions in PDPI result predominantly from neutral rather than ionic decomposition. PDPI should exhibit high sensitivity,since both dissociation 0 1990 American Chemical Society
