Anal. Chem. 2001, 73, 606-611
Determination of Low Molecular Weight Silicones in Plasma and Blood of Women after Exposure to Silicone Breast Implants by GC/MS Daniela Flassbeck, Bettina Pfleiderer,† Rainer Gru 1 mping, and Alfred V. Hirner*
Institute of Environmental Analytical Chemistry, University of Essen, 45177 Essen, Germany and Institute of Clinical Radiology, Westfalian-Wilhelms University, Muenster, Germany.
A sensitive, one-step sample preparation method for detection of volatile, low molecular weight (LMW) cyclic silicones hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), and dodecamethylcyclohexasiloxane (D6) in plasma and blood using gas chromatography coupled with mass spectrometry (GC/MS, SIM mode) is presented. In spiked experiments, extraction efficiencies for these siloxanes (100-20 000 ng/mL) were approximately 90% for plasma and approximately 80% for blood; only in the case of D3 was the recovery very low. Plasma and blood of women who are or were exposed to silicone gel-filled implants and of control subjects were analyzed for low molecular weight silicones. D3-D6 were not detectable in control plasma or blood. Although the investigated numbers of patients samples are very limited, and thus, no statistical analysis is possible, our data clearly show a general increase in the amount of LMW cyclic siloxanes in the bodies of women with silicone implants. In particular, several years after ruptured silicone implants were removed, siloxanes could still be found in blood samples from several women. Siloxane compound D3 varied between 6 and 12 ng/mL (plasma) and between 20 and 28 ng/mL (blood), whereas the concentration range of D4 was 14-50 ng/mL (plasma) and 79-92 ng/mL (blood). D5 and D6, with one exception, could not be detected. Poly(dimethylsiloxane) has been widely used in consumer applications such as personal care and biomedical products, including silicone gel-filled breast implants for breast augmentation and breast reconstruction following mastectomy, over the last three decades. The gel of silicone gel-filled breast implants contains only 1-2% of low molecular weight (LMW) silicones, with structures identified mainly as cyclic compounds such as hexamethylcyclotrisiloxane (D3), octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), dodecamethylcyclohexasiloxane (D6), and tetradecamethylcycloheptasiloxane (D7). The other 98% of silicone gel is composed mostly of high molecular weight (HMW) silicones * Corresponding author. Phone: +49 (201) 183-3950. Fax: +49 (201) 1833951. E-mail:
[email protected]. † Westfalian Wilhelms-University.
606 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
that cannot be separated using GC with a capillary column.1 D5D7 are the main LMW silicones in silicone implant gel.1 Since 1978, it has often been reported that silicone can “bleed” from clinically intact implants and can be detected visually in lymph nodes.2-4 Under laboratory conditions, the release of D4D7 from intact silicone implants into a lipid-rich medium occurs more readily than the release of the other LMW silicones migrating from the gel.1 The observed accumulation of siloxanes in various organs of mice following subcutaneous injection of D3D7-containing breast implant distillate and the biodegradation of injected siloxanes in lymph nodes of rats confirmed these early results.5,6 1H NMR localized spectroscopy was used to detect migration and accumulation of silicone in the livers of women with silicone gel-filled breast implants.7 All of these results indicated that leaked silicone is not limited to tissue surrounding the implant; there may also be transport by the circulatory and lymphatic systems. During the past few years, several element-specific methods for the investigation of silicon in biological substances were developed.8-10 It is still disputed whether the measured levels of silicon in serum or plasma correlate with the LMW silicones leached from implants. Consequently, it is of importance to analyze the LMW contaminants in biological materials. Several studies have focused on the extraction of LMW silicones from biological matrixes with the use of various chromatographic techniques such as HPLC, GC/AED, and GC/MS.5,11-13 The siloxane recoveries (1) Lykissa, E. D.; Kala, S. V.; Hurley, J. B.; Lebovitz, R. M. Anal. Chem. 1997, 69, 4912-4916. (2) Barker, D. E.; Retsky, M. I.; Schultz, S. Plast. Reconstr. Surg. 1978, 61, 836-841. (3) Nelson, G. D. Plast. Reconstr. Surg. 1980, 66, 969-970. (4) Hauser, R J.; Schoen, F. J.; Pierson, K. K. Plast. Reconstr. Surg. 1978, 62, 381-384. (5) Kala, S. V.; Lykissa, E. D.; Neely, M. W.; Lieberman, M. W. Am. J. Pathol. 1998, 152, 645-649. (6) Pfleiderer, B.; Moore, A.; Tokareva, E.; Ackerman, J. L.; Garrido, L. Biomaterials 1999, 20, 561-571. (7) Pfleiderer, B.; Garrido, L. Magn. Reson. Med. 1995, 33, 8-17. (8) Peters, W.; Smith, D.; Lugowski, S.; McHugh, A.; Baines C. Ann. Plast. Surg. 1995, 34, 343-347. (9) Jackson, L. W.; Dennis, G. J.; Centeno, J. A. In Metal Ions in Biology and Medicine, Collery, Ph., de Braetter, V. N., Khassanova, L., Etienne, J. C., Eds.; John Libbey Eurotext: Paris, 1998; Vol 5; 33-38. (10) Kennan, J. J.; McCann Breen, L. L.; Lane, T. H.; Taylor R. B. Anal. Chem. 1999, 71, 3054-3060. (11) Kala, S. V.; Lykissa, E. D.; Lebovitz, R. M. Anal. Chem. 1997, 69, 12671272. 10.1021/ac000738z CCC: $20.00
© 2001 American Chemical Society Published on Web 12/22/2000
Table 1. Extraction Efficiencies of LMW Silicones in Spiked Human Plasma sample vol (mL)
spiked plasma conc (ng/mL)
D3 (m/z 209)
0.5 1 0.5 1 0.5 1
200 100 2000 1000 20 000 10 000
32 ( 1 28 ( 1 21 ( 2 16 ( 1 20 ( 1 25 ( 2
from mouse liver homogenates spiked with varying amounts of PDMS-V, containing linear and cyclic poly(dimethylsiloxane), extracted with ethyl acetate and analyzed by GC/MS and GC/ AED, were found to be greater than 90%.11 A study for detecting radioactively labeled D4 and its metabolites from plasma and blood revealed a concentration range between 21 and 2100 µg/mL; a lower range between 50 and 500 ng/mL was found by extracting blood in the presence of glass beads.12 After single extraction of these materials with THF, the efficiency was 79-92% for the higher concentrations, depending on the material, and 82-102% for the lower concentration range; however, the monitoring of siloxanes in real biological samples by this analytical technique works well only if the silicone is radioactively labeled. Nevertheless, the sample preparation method has been used to measure D4 in the plasma of human volunteers exposed to D4 vapor.13 The goal of this work was to develop a very sensitive, onestep sample preparation method with very low solvent volume, high sensitivity, and reproducibility to detect cyclic LMW silicones in human plasma and blood, especially D4-D6, released from silicone gel-filled breast implants. First, the extraction efficiencies for D3-D6 in spiked plasma and whole blood were determined. The developed method was used to investigate plasma and blood samples of women who are or were exposed to silicone prostheses, as well as samples from control subjects. We were able to detect siloxanes only in the plasma or blood of women who are/were exposed to silicone gel-filled implants. EXPERIMENTAL SECTION Reagents and Chemicals. All cyclic siloxanes (D3-D6) with purities ranging from 95 to 97% and the internal standard tetrakis(trimethylsiloxy)silane (M4Q) were obtained from Gelest Inc. (Karlsruhe, Germany), while hexane was obtained from Merck KGaA (Darmstadt, Germany, SupraSolv). Siloxanes and the internal calibration standard were dissolved in hexane. The siloxane-spiked solutions and internal standard solution (10 µg/ mL) for sample preparation were dissolved in hexane, and the last solvation step was done in methanol. Measurement of Siloxanes. Gas chromatography/mass spectroscopy (GC/MS) analysis of siloxanes was performed with a Hewlett-Packard 5890 Series II gas chromatograph equipped with a HP 7673 and coupled to a HP 5989 A MS engine. Data analysis was carried out using HP-UX MS Chem Station software. The GC/MS conditions were as follows: injector port temperature, (12) Varaprath, S.; Salyers, K. L.; Plotzke, K. P.; Nanavati, S. Anal. Biochem. 1998, 256, 14-22. (13) Utell, M. J.; Gelein, R.; Yu, C. P.; Kenaga, C.; Geigel, E.; Torres, A.; Chalupa, D.; Gibb, F. R.; Speers, D. M.; Mast, R. W.; Morrow, P. E. Toxicol. Sci. 1998, 44, 206-213.
extraction efficiencies (%) D4 (m/z 281) D5 (m/z 355) 102 ( 4 96 ( 1 91 ( 2 87 ( 1 83 ( 1 90 ( 2
87 ( 3 85 ( 2 100 ( 1 99 ( 1 98 ( 2 89 ( 3
D6 (m/z 341) 105 ( 2 112 ( 3 93 ( 2 95 ( 1 104 ( 3 93 ( 3
200 °C; initial oven temperature, 40 °C for 2 min; initial ramping rate, 10 °C/min to 160 °C, followed by a second ramping rate of 30 °C/min to 320 °C, which was subsequently held for 5 min. Siloxanes were separated on a HP 5 MS column (5% phenylmethylsilicone stationary phase from Hewlett-Packard with column dimensions of 0.25 mm × 30 m and 0.25 µm film thickness). Standards, extracts of the spiked samples, and real samples were run in the SIM mode (using quantifying fragments for D3-D6 and internal standard m/z 207, 281, 267, 355, 341, 429, and 281) using EI (electron impact) for ionization. Siloxanes lose a methyl group in the EI mode and generate ions of molecular mass minus 15 mass units (M-15). The measured concentrations were calculated by the HP-UX Chem Station software with internal standard calibration. Extraction of LMW Silicones from Spiked Plasma, Blood, and Water. All of the spike experiments for determining the extraction efficiencies of siloxanes were performed with 0.5- and 1.0-mL sample volumes in 2.5- and 10-mL vials. 100 µL of methanolic multisiloxane standard solution with concentrations of 1, 10, and 100 µg/mL and 100 µL of M4Q solution were added to each sample. The spiked samples were extracted with 1 mL of hexane, then vortex-mixed with a Vortex Genius II at high speed setting for 5 min and centrifuged afterward in a Sigma 302 centrifuge at 10 000 rpm for 20 min. Each concentration experiment was repeated 3 times to establish reproducibility. Extraction of Plasma and Blood from Women with Silicone Gel-Filled Implants and from Control Subjects. The 10 plasma samples of the patient group were stored at 3 °C in the dark for nearly one year, and the four patients' blood samples were stored at -20 °C for three months. The storage conditions of the six plasma and two blood control samples were the same as for the patients’ samples. Plasma (separated from blood by centrifugation at 2000g) and blood samples were spiked with 100 µL of M4Q as internal standard. Sample preparation was conducted as described above. Each sample extract was analyzed 3 times. The ages of the subjects ranged between 38 and 73 years for the patients, and between 24 and 38 years for the control group. The silicone breast implants are/were implanted for 5-30 years (mean, 14 years). Seven patients had undergone explantation of their respective prostheses after blood was sampled (mean time after implant removal, 4.1 years). The status of the implants was determined at the time of implant removal. The patients’ characteristics are summarized in Table 3 and Table 4. RESULTS AND DISCUSSION A typical chromatogram of a multi siloxane standard solution that was obtained by GC/MS in the sensitive SIM modus is Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
607
Table 2. Extraction Efficiencies of LMW Silicones in Spiked Human Blood sample vol (mL)
spiked blood conc (ng/mL)
D3 (m/z 209)
0.5 1 0.5 1 0.5 1
200 100 2000 1000 20 000 10 000
4(1 5(2 10 ( 1 10 ( 1 10 ( 1 10 ( 1
extraction efficiencies (%) D4 (m/z 281) D5 (m/z 355) 104 ( 2 97 ( 4 105 ( 1 93 ( 1 92 ( 1 77 ( 1
79 ( 2 64 ( 4 89 ( 1 70 ( 2 87 ( 2 71 ( 1
D6 (m/z 341) 89 ( 3 90 ( 5 77 ( 1 66 ( 3 87 ( 3 74 ( 31
Table 3. Cyclic Siloxanes in Plasma of Women Who Currently Have or Previously Had Silicone Gel-Filled Implants conc (ng/mL) years
statusa
diagnosis
D3 (m/z 209)
D4 (m/z 281)
D5 (m/z 355)
D6 (m/z 341)
8 10 10 30 5
in situ in situ in situ in situ explanted 5 years prior
2(1 10 ( 1 12 ( 1 8(1 nd
2(1 32 ( 1 36 ( 1 14 ( 1 3(2
nde nd nd nd 28 ( 1
nd nd nd nd 17 ( 1
6 13 15 20
explanted 2 years prior explanted 5 years prior explanted 4 years prior explanted 5 years prior
6(1 nd nd 6(1
20 ( 1 nd nd 50 ( 1
nd nd nd nd
nd nd nd nd
24
explanted 4 years prior
intact intact (bi)b intact (bi) intact (bi) ruptured (rb)c gel bleed (lb)d ruptured (bi) bleed (bi) intact ruptured (rb) intact (lb) intact (bi)
nd
nd
nd
nd
a
Implantation status when blood was sampled. b bi, both implants. c rb, right breast. d lb, left breast. e nd, not detected.
Table 4. Cyclic Siloxanes in Whole Blood of Women Who Currently Have or Previously Had Silicone Gel-Filled Implants years of implant
a
statusa
12
in situ
13 13
in situ in situ
22
explanted 4 years prior
diagnosis
D3 (m/z 209)
(rb)c
ruptured intact (lb)d intact (bi)b ruptured (rb) intact (lb) ruptured (bi)
conc (ng/mL) D4 (m/z 281) D5 (m/z 355)
D6 (m/z 341)
nde
nd
nd
nd
28 ( 6 nd
79 ( 1 nd
nd nd
nd nd
20 ( 1
92 ( 1
nd
nd
Implantation status when blood was sampled. b bi, both implants. c rb, right breast. d lb, left breast. e nd, not detected.
presented in Figure 1. D3 and D4 have only one main mass fragment (m/z 207 and 281, respectively) each that can be used for quantification, but D5 (m/z 73, 267, 355) and D6 (m/z 73, 341, 429) have three mass fragments each that are possibly suited to this purpose. Criteria for the mass selection are a high characteristic identification value for the compound (high mass fragment) and a high abundance of the fragment (high sensitivity). While the high molecular mass fragments of D5 exhibit almost the same respective abundances, the intensity of D6 fragment m/z 341 is slightly higher than the one of m/z 429. When measuring the extracts from all samples that are based on both alternative fragments, the concentrations were nearly the same for D5 and D6. To simplify matters, we demonstrate here data for only one mass fragment (D5, m/z 355; D6, m/z 341). Our detection limit for each of D3-D6 is 2 pg/µL, which is similar to values reported in the literature.14 In the literature, THF and ethyl acetate are suggested for successful extraction of poly(dimethylsiloxane). These solvents (14) Varaprath, S.; Lehmann, R. G. J. Environ. Polym. Degrad. 1997, 5, 17-31.
608 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
are miscible with water and should lead to high extraction efficiencies;12 however, trace quantities of D3-D6 were detected in blanks for these two solvents when analyzed in the SIM mode, while one of the most common GC solvents, hexane, did not show any evidence of contamination by direct injection. This was also true for hexane after passing the sample preparation in vials used for extraction and centrifugation. In addition, because hexane is immiscible with water, which is contained in biological samples, no drying step is necessary before GC/MS analysis. Therefore, hexane seemed to be the most suitable solvent for the extraction of low quantities of siloxanes in biological material. Hexane blanks were run between samples to avoid possible carry-over effects and to check the blank levels, but no artifacts were seen. Extraction Efficiencies of Cyclic Siloxanes from Plasma, Blood and Water. To test the extraction efficiency for D3-D6, we spiked plasma, whole blood, and water with concentrations between 100 and 20 000 ng/mL. At the beginning of the study, we used 2.5-mL vials for extracting plasma samples. In most of the experiments, we found
Figure 1. GC/MS analysis (SIM mode) of a multisiloxane standard containing hexamethylcyclotrisiloxane (D3, [3.5 min]), octamethylcyclotetrasiloxane (D4, [6.5 min]), decamethylcyclopentsiloxane (D5, [9.2 min]), internal standard tetrakis(trimethylsiloxy)silane (M4Q, [9.7 min]) and dodecamethylcyclohexasiloxane (D6, [11.7 min]).
extraction efficiencies for D4-D6 in the range of 45-70% (data not given here); only the investigation of high concentration levels (10 000 ng/mL) yielded efficiencies approaching 90%. To improve the accuracy of the results, other authors repeated extractions 3 times with fresh solvent and combined the individual extracts, resulting in a large extract volume.12 However, when working with low analyte quantities in samples, it is necessary to concentrate these large volumes, and there is a risk that solvent and some of the extracted siloxanes may also be volatilized inadvertently. To overcome this potential problem, a different strategy was adopted. The use of 10-mL vials improves mass transport, thus resulting in higher recovery efficiencies for D4-D6. A concentration step is no longer necessary, and the loss due to analyte volatility is significantly reduced. The extraction efficiencies were found to be greater than 80% (Table 1). There was no significant reduction in efficiency for lower concentrations at the analyte levels studied. The recovery of D4 was lower than that for D5 and D6 due to its higher volatility. In all of the spike experiments, the extraction efficiency of D3 was determined to be very low in comparison to the other siloxanes. The vapor pressure of volatile D3 is 12.9 mmHg, and its solubility in water of 1.57 mg/L is the highest of the cyclic siloxanes under investigation.15 The relatively hydrophobic D3 could, therefore, accumulate at the surface of the sample and volatilize before treating the sample with hexane. Because of its insolubility in water, hexane is probably not the most appropriate solvent for extraction of this compound. Neither ethyl acetate nor THF could be used due to their high blank values. Our data indicate that the investigated siloxane species, with the exception of D3, can be recovered quantitatively from plasma. Increasing the amount of hexane that is used in the extraction from 1 mL to 2 mL did not increase the extraction efficiencies for (15) Van Post, D. C. Water Pollut. Control 1978, 77, 520-524.
cyclic siloxanes (not shown). The sample volume had no statistically significant influence (tested with paired t test) on the extraction efficiencies (Table 1). Plasma is only one of several blood fractions or components that could possibly contain siloxanes, and therefore, we expanded our research to whole blood. The same concentration range and 10-mL vials, as in the plasma study, were used for blood samples. The extraction efficiencies for D4-D6 were around 80% (Table 2). There was no statistically significant reduction of the recovery efficiencies for low concentrations, and even the comparison between plasma and blood did not show statistically significant differences in extraction efficiencies. This was unexpected because the composition of blood is much more complex than that of plasma. One may hypothesise that siloxanes could be adsorbed on the various blood components, resulting in a less efficient extraction. Our data revealed, however, that D4-D6 are extractable and, furthermore, retained the same high yields. There is only one comparative study dealing with the recovery of D4 and its metabolites from plasma, blood, and various other biological samples.12 For a single extraction, the recovery of D4 from plasma was found to be around 90%, which is similar to our results for D4-D6 in plasma. Our efficiencies for the recovery of these compounds from blood were slightly higher: in the high concentration range, Varaprath and co-workers found efficiencies around 80% after a single extraction.12 When dealing with low concentrations, this group found it necessary to use glass beads to get a recovery efficiency for D4 of around 90% after a single extraction; however, we did not find any differences in the recovery rates, even when using low concentrations of siloxane. It was not necessary to use glass beads when determining low analyte concentrations due to our use of larger vials and, thus, improved mass transport for extraction. When comparing results for plasma with those for blood, it can be seen that the efficiencies in plasma are slightly better, but not statistically significant, as compared to those for blood. Exploring the same concentration range in water, recoveries of the siloxanes were found to be dependent on the concentrations that were examined. The most favorable results were found in the 10 000 ng/mL samples, with greater than 80% efficiencies for D4-D6. In the lower concentration range, the recovery rates were around 40% which indicates that the high volatility of the siloxanes leads to sample loss in hydrophilic samples (data not shown). Extraction of Plasma and Blood Samples from Women Who Currently Have or Previously Had Silicone Breast Implants. The new method was applied to examine both plasma and blood from women with silicone gel-filled implants, women who had undergone explantation, and control subjects. Ten plasma samples and 4 blood samples from women with breast implants (n ) 7) or after explantation (n ) 7) and 6 plasma samples and 2 blood samples of control subjects were measured. The patients’ results are summarized in Table 3 and Table 4. Even though silicone is used in the production of plastic products, and most medical devices are coated with silicone, no cyclic siloxanes (D3-D6) could be found in plasma of the control group, with detection limits of 2 pg/µL. We could see humps in the chromatograms that corresponded to the retention peaks of these compounds, which indicated the possible presence of siloxanes in very low quantities. In contrast, D3 and D4 could be Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
609
Figure 2. Analysis of a plasma extract of a 62-year-old women whose 5-year-old silicone gel-filled breast prostheses were removed 5 years prior. One implant was ruptured at the time of surgery, and the other was bleeding. We could detect D4-D6 [6.5 min, 9.2 min, 11.7 min] in a concentration range between 3 and 28 ng/mL plasma using GC/MS with tetrakis(trimethylsiloxy)silane [9.7 min] as internal standard. At retention times greater than 19 min, three signals were found while monitoring m/z 341. We do not know if these signals belong to higher molecular weight silicones.
detected in 7 of 10 plasma samples and in 2 of 4 whole blood samples of patients who had silicone implants in situ or had their silicone implants removed at the time when blood sampling was done (Tables 3 and 4). In plasma samples, D3 was found in very low concentrations, which is in agreement with the results of our spiked experiments. Due to the volatility of D3, a high loss rate for this compound must be considered during sampling and sample preparation. Furthermore, D3 has the lowest concentration of the cyclic siloxanes in the implant gel.1 In most cases (7/10), only D4 was detected in plasma at levels lower than 50 ng/mL. Only in one plasma sample, from a woman who had defective implants, was the determination of the three siloxanes D4, D5, and D6, possible (Figure 2). Compounds D3 and D4 could be detected in plasma samples of women who had silicone implants in situ which subsequently were determined to be intact intraoperatively. There was no significant correlation between implantation time and siloxane concentration. Even though the implant shells were macroscopically intact, siloxanes could conceivably bleed out of the shell and migrate into the blood. In contrast, plasma samples of two women with supposedly intact implants had no detectable siloxanes after explantation of their silicone implants four and five years later, respectively. Furthermore, no siloxanes could be detected in the plasma of a woman whose implants exhibited signs of gel “bleed” at the time of implant removal (five years previously). These findings indicate that concentrations of siloxanes circulating in blood are below our detection limit, because siloxanes probably leave the body via the lungs or kidneys, or possibly accumulate in organs over time, as reported by Kala et al. in an animal study.5 D3 and D4, and in one case D4-D6, could be measured in the plasma of women who had ruptured implants removed. 610 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
Figure 3. Analysis of a whole blood extract of a 49-year-old woman who had two silicone implants for 22 years, which were subsequently explanted 4 years prior and classified to be ruptured. D3 [6.5 min] and D4 [9.2 min] could be detected by GC/MS in concentrations of 20 and 92 ng/mL, respectively, by using tetrakis(trimethylsiloxy)silane [9.7 min] as internal standard. For D5 and D6, humps could be seen where the expected peaks would normally occur.
The number of whole blood samples of women with prostheses was limited; nevertheless, D3 and, in particular, D4 were found in half of the investigated blood samples (2/4) and in higher concentration than in plasma samples. For D4, the concentration was around 80 ng/mL (Table 4). Three of four women whose whole blood was measured had silicone gel-filled implants in situ at the time of blood sampling. Siloxanes could be detected in the blood of one woman who had intact implants. In the blood samples of the other two women, it was not possible to detect siloxanes, even though one implant of each subject was ruptured. Because bleeding of intact silicone breast implants is known and we had found compounds D3 and D4 in plasma when implants were supposedly intact, it seems surprising and even contradictory that we could not detect siloxanes in the blood of women in which rupture was confirmed for one of the two implants. Possible reasons for these results could be either a very low bleeding rate of the implant, or the occurrence of the implant defect after blood sampling, perhaps during removal. A further possibility is that the siloxane transport from the tissue surrounding the implant into the blood was delayed until after the sampling event. Implant removal was performed 6 and 13 months after drawing of blood, respectively. Siloxanes D3 and D4 could be found in the whole blood of a woman whose ruptured implants were removed 4 years prior. The higher siloxane amounts in whole blood, as compared to those detected in plasma, could be explained by the complete and undivided blood sample: first, during centrifugation of the whole blood to separate the plasma, siloxanes could be volatilized and thereby removed from the investigation. Furthermore, the siloxanes are probably not limited to the plasma fraction.16 When (16) Kossovsky, N.; Heggers, J. D.; Robson, M. C. J. Biomed. Mater. Res. 1987, 21, 1125-1131.
comparing plasma samples to whole blood samples with nearly the same implantation time (10 and 13 years), the detected siloxane levels in whole blood were much higher than the corresponding levels in plasma. This is even more remarkable given the lower extraction efficiencies for D3-D6 from whole blood. Siloxanes were detected in blood samples of women who had undergone surgery to remove their broken prostheses and in women who still had implants in place that are considered to be intact. In most of these samples, D3 and D4 were found. In a laboratory study, these siloxanes were released from intact implants in relatively high concentrations into air;1 nevertheless, D4 was the second most abundant siloxane detectable in the lipidrich medium. In humans, these volatile siloxanes could be lost via the lungs, other membrane surfaces or pores, or may even accumulate in different organs,6 but the dynamics of these processes are still unknown. In an experiment described by Utell et al.,13 volunteers inhaled 10 ppm D4 (122 µg/L) via a mouthpiece during a 1-h exposure. The preexposure and 24-h postexposure levels of D4 vapor in plasma of human subjects who were exposed to this siloxane were not significant, according to the limit of their quantitation criteria, but the mean value was significantly greater than zero.13 Because we could measure D3 and D4 in the patient group whose prostheses were removed, it is possible that our quantitation criterion was lower than that for D4 of the other research group. On the other hand, quantification of siloxanes was also possible in two of four blood samples of women with silicone breast implants, even though we could see a slight hump in the chromatograms of the negative samples. In the literature, there are various studies by other groups who estimated silicon levels in blood and blood products of volunteers without implants, by GC/MS. The detection levels varied between 10 ng/mL and 5 µg/mL.17 This wide range could be explained by inaccuracies in analytical performance, differences in analytical methods, and different nutritional habits in various geographical regions.17 (17) Lugowski, S. J.; Smith, D. C.; Lugowski, J. Z.; Peters, W.; Semple, J. Fresenius J. Anal. Chem, 1998, 360, 486-488.
We calculated silicon values based on our analyzed cyclic volatile siloxanes of 2-21 ng/mL in plasma and ∼35 ng/mL in blood of women who are or were exposed to silicone implants. Our calculated plasma and blood silicon values of patients with silicone prostheses are in the lower range of the accepted total “normal” silicon amount of control subjects.17 Therefore, it is not possible to see significant differences between patients with silicone implants and controls when only investigating total silicon values as described in the literature. CONCLUSIONS In several studies, no differences were found between the blood silicon concentrations of women with silicone breast implants and controls.9,17 Our results, however, clearly indicate that there is a relationship between exposure to silicone gel-filled implants and LMW cyclic siloxane contamination in plasma. In the case of the results of the four investigated blood samples in the ratio of 1:1, no relationship could be discerned between implants and siloxanes. Even though the investigated numbers of plasma and blood samples are very limited, and thus, no statistical analysis was possible, our data clearly show a trend that implantation with silicone prostheses increases the amount of LMW cyclic siloxanes in the body. In particular, siloxanes could still be detected in blood samples from several women some years after broken silicone implants were removed. When intact implants were removed, no siloxanes could be found. Our methodology is useful when analyzing biological materials for mobile LMW cyclic siloxanes which could accumulate in different organs. ACKNOWLEDGMENT The authors thank the Forschungspool of the University GHS of Essen for financial support of this work.
Received for review June 27, 2000. Accepted October 25, 2000. AC000738Z
Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
611
