Analyst, June 1996, Vol. 121 (53R-63R) 53R Silicones and Their Determination in Biological Matrices A Review Biljana A. CaviC-Vlasaka*, Michael Thompsona* and Dennis C. Smithb a Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 1 A1 , Canada Ontario M5S IA4. Canada Centre for Biomaterials, University of Toronto, I70 College Street, Toronto, Summary of Contents Introduction Properties and Applications of Silicones Biochemistry of Elemental Silicon and Silicones General Approach to the Determination of Trace Amounts of Survey of Methods Used for Non-specific and Silicones in Biological Materials Silicon-specific Microstructural Determination of Silicones in Tissues (Microanalysis) Silicon-specific (Elemental) Techniques for the Detection of Silicones in Biological Materials Survey of Silicone-specific Techniques IR, FTIR and Raman Spectrometry NMR Spectrometry GC and GC-MS Methods Silicone Biotransfonnation Products in Biological Samples Problem of Speciation of Silicon and Identification of Conclusions References Keywords: Silicones; trace analysis; biological samples; biotransformation; review Introduction In recent years, a revolution has taken place in the biological sciences in terms of the transition from relatively macroscopic research to enormous advances in molecular biology.These advances have been driven, in part, by the development of techniques such as multi-dimensional NMR, high-resolution DNA sequencing, routine high throughput of structures by charge-coupled device X-ray crystallography, polymerase chain reaction technology, recombinant DNA methods and an array of other methods.Such advances have led to an expansion in the generation of biologically derived products. These entities have presented tremendously challenging tasks for the analytical chemist in terms of the identification and the determination of species present in complex biological mat- rices. The purity of protein-based drugs (and the necessary regulatory baggage that is associated with these moieties) and analysis of fermentation broths are just two examples in this area. Another aspect of the analytical chemistry of biological matrices which presents almost overwhelmingly difficult prob- lems for the analytical scientist is the examination of specific tissues in relation to the deliberate placement of devices in the human body.In this connection, the use of modern biomaterials spans a large range of structures from artificial teeth, veins and * To whom correspondence should be addressed. knees to heart pacemakers and the like. The number of materials being employed for the fabrication of these devices is also large, from metals such as titanium to polymers such as polyurethane. One species that has been used extensively in vivo in recent times is silicone. For example, this material is utilized in the construction of catheters and mammary and facial implants. Among the most widely used devices have been breast prostheses, which consist of a silicone elastomer envelope containing a silicone gel. The latter is typified as 25% of a high relative molecular mass (300 000400 000 Da) cross-linked polysiloxane matrix containing 75 % of low relative molecular mass (6000-19 000 Da) polydimethylsiloxane fluid.More than 1000000 of these breast implants have been placed in women in the USA and more than 100000 in Canadian women. Experimental and clinical evidence has accumulated to show that silicone elastomers in vivo shed particulate matter that may provoke an inflammatory response. Silicone gel-filled implants may release silicone fluid also by diffusion through the silicone elastomer envelope or by frank rupture of the envelope. Fibrosis and (in the long term) calcification in response to these sequelae have been observed. A variety of other clinical complications have also been attributed to silicone implants, including immune reactions, specifically various types of connective tissue disease. Considerable controversy exists as to the nature and magnitude of these effects.A major problem in the evaluation of clinical effects of inorganic and organic forms of silicon is the lack of information on ‘normal’ ranges of silicon systemically and locally in the body. Further, few mechanistic studies have been carried out on the migration, transfer, biodegradation and excretion of sili- cones. There are many gaps in our knowledge of silicon biochemistry and few reliable studies of, for example, silicon concentrations in blood and serum. Because of the ubiquity of silicon and silicones, trace element analysis for silicon requires a particularly careful approach with stringent precautions. The analysis of tissues for silicones is also problematic because of their potential fugitivity and volatility.The magnitude and importance of the clinical questions related to silicon and silicones demand a definitive approach to the available analytical procedures. In this review, we present a perspective on analysis in biological matrices. Properties and Applications of Silicones The term silicone, first used by Kipping in 1901 ,2 is a generic name for polymers based on organosilicon chemistry and the polysiloxanes, containing the Si-0-Si backbone in general. The most important siloxane polymers are polydimethylsiloxanes (PDMS), [-Si(CH&O-],. PDMS exist as linear polymers with degrees of polymerization varying from 0 (hexamethyldisilox- ane) to more than 1000, and as cyclic chains or rings.A variety54R Analyst, June 1996, Vol. 121 of other silicone polymers are produced by replacing the methyl groups with different organic substituents. Trifunctional molec- ular siloxane species bear potential cross-link sites producing branching within the molecule. Depending on the degree of polymerization and cross-linking pattern, silicone materials could be of fluid, gel, gum (elastomer or rubber) or resin type. PDMS are generally considered to be chemically and physiologically inert materials. Although they are recognized as compounds which are stable to thermal and oxidative degrada- tion, their thermal rearrangements are possible in the presence of both acids and bases. A thermodynamic equilibrium mixture of PDMS contains approximately 88% of linear and 12% of cyclic siloxane components at temperatures up to 200 "C.3 The PDMS backbone is the structural component for one of the most flexible chain molecules, both in the dynamic (ability of molecule to change spatial arrangements by rotations around its skeletal bond) and in the equilibrium sense (ability of a chain to be compact when in the form of a random coil).The most important reasons for this flexibility lie in the remarkable length of the Si-0 bond (compared with the C-C bond) and in the particular openness of the Si-0-Si bond angle (in comparison with the usual tetrahedral bond). High rotational and oscillatory freedom of the methyl side groups and low intermolecular interactions are considered to be the origin of some extraordi- nary properties of PDMS, such as low values for characteristic pressure, bulk viscosity (q), entropies of dilution and excess volumes on mixing PDMS with solvents. In contrast, PDMS possess remarkably high permeability to gases.4 Silicones possess excellent ability to modify surfaces and interfaces.The low liquid surface tension of PDMS (16-21 mN m-1 at room temperature depending on molecular weights) accounts for their tendency to accumulate at air-substrate surfaces. This feature is associated with the flexibility of the siloxane bond, freedom of methyl groups and the high energy and partial ionic nature (41% ionic bond contribution) of the siloxane bond. The partially ionic siloxane backbone is susceptible to hydrolysis by water at extremes of pH.3,5,6 General structures for, and some of the properties of, some linear and cyclic siloxanes are shown in Fig.1 and Table 1. Most of the physical properties of PDMS increase on insertion CH, r 7~~ 1 CH, (4 I I CH3- Si - 0 - Si - 0 - Si - CH, 1 1 1 1 1 - 0 - S i - 0 - r r"' 1 L CH3 Jn Fig. 1 siloxanes. General structure of (a) linear (MDnM) and (b) cyclic (D,) of dimethylsiloxane units. The viscosity of highly polymerized PDMS could be as high as several million centistokes. Whereas hexamethyldisiloxane is considered to be a very volatile compound (Table l), highly polymerized PDMS are distin- guished by their involatility. The best solvents for all PDMS fluids are aromatic and aliphatic hydrocarbons and chlorinated solvents.Highly polymerized PDMS are incompatible with mineral oils and alcohols. Silicones are excellent dielectrics and provide a very high electric impedance. Silicones are recog- nized by excellent mechanical durability but at the same time they are materials of weak tensile strength.9 Although silicon is an important trace element for plants and animals, playing an indispensable role in the biosphere, organosilicon compounds have not been positively identified as naturally occurring. There is no conclusive evidence that organic compounds in which silicon is bound to oxygen or nitrogen occur in nature. Also, naturally occurring organosili- con compounds containing silicon+arbon linkages have not been unequivocally detected in natural systems.3JOJ 1 However, man-made organosilicon compounds, mainly siloxanes, are widely dispersed throughout the environment.Silicones are the hydrolysis and polymerization products of organochlorosilanes, which are prepared from silica. Silica is heated and reduced to silicon, which is treated with methyl chloride in order to produce chlorosilanes. Dimethyldichlorosilane as a major monomer forms dimethylsilanediol when treated with water. This last, unstable compound condenses into siloxanes.2 Since they were first commercially introduced in the USA in 1943,2 the production and application of silicones has grown rapidly so that it was estimated in 1990 that some 700 000 tons of silicones are produced annually.12 The properties of silicones described briefly above yield the basis for their very wide application.Silicone materials are applied in a variety of fields. Some of their most common applications are their uses as adhesives, protective coatings, caulking materials, encapsulation media, high-performance elastomers, membranes, water repellents, surfactants, anti- foaming agents, lubricants, polishes, printing inks, textile finishes, hydraulic, heat-transfer and dielectric fluids, electrical insulators, organic syntheses aids, liquid phases for gas chromatography, silylation agents, release control agents for agricultural chemicals, et~..3,479,~~ Being non-toxic and mainly non-irritating, silicones are also applied in the cosmetic industry during the fabrication of shampoos, hair conditioners, per- spirant formulations, sunscreen formulations and different make-up products (the high refractive index of silicones contributes to the gloss of products).14 Some biologically active siloxanes (e.g., 2,6-cis-diphenyl- hexamethylcylcotetrasiloxane) find practical application as therapeutic agents in medicine and as reference drugs in experimental pharmacology.Silicones are also applied as drug release and delivery compounds.lO Silicones that are distinguished by high-purity grade are called medical-grade silicones and, in this form, can be applied clinically. As biocompatible materials (non-rejection by tissue is claimed)l5 with minimal reactivity with biological systems, Table 1 Properties of some linear and cyclic siloxanes Refractive index, nD25 Compound (ref. 7) Hexamethyldisiloxane (MM) 1.3748 Octamethyltrisiloxane (MDM) 1.3822 Decamethyltetrasiloxane (MD2M) 1.3872 Octamethylcyclotetrasiloxane (D4) 1.3940 Decamethylcyclopentasiloxane (D5) 1.3957 Density (25 "C)/g cm-3 (ref. 7) 0.7619 0.8200 0.8536 0.950 0.9528 Viscosity (25 OC)/cS (ref.7) 0.65 1.04 1.53 2.30 3.87 Vapour pressure BpPC (25 "C)/mmHg (ref. 7) (ref. 8) 100.5 41.73 152.5 - 196.4 - 175.4 0.85 211.0 -Analyst, June 1996, Vol. 121 55R they have been in some way used in almost all surgical fields. Inertness, stability and pliability of silicones make them important devices in cardiovascular surgery as parts of tubing and membrane systems incorporated in cardiovascular devices such as by-passes and pacemakers. The same properties have been the reason for their application as orthopaedic implants (silicone-rubber joints), in facial reconstruction and as mam- mary and penile implants.They are also parts of drainage systems and catheters. Silicone artificial skin and contact lenses take advantage of the high permeability of these materi- The silicone breast implant is the subject of tremendous current controversy. There are estimations that almost 1 million women have used them either for cosmetic or reconstruction purposes over three decades.17.18 The question of the safety of breast implants and their possible adverse effects on health is the subject of numerous discussions. The basic structure of silicone breast implants is a silicone rubber (elastomer) envelope filled with silicone gel or saline. The envelopes are sometimes coated with polyester polyurethane.The reinforce- ment of silicone rubber is performed by the introduction of fine particles of silica. Silicone gel is a mixture of relatively low and high molecular mass PDMS components.19.20 als.9,15.16 Biochemistry of Elemental Silicon and Silicones Silicon is one of the most prevalent elements on our planet. Most living organisms contain at least trace levels of silicon and some primitive organisms even have a silicate skeletal structure. In biological systems, as well as in the whole environment, silicon occurs in its inorganic form mainly as silica, silicic acid and silicates, but it can be specifically associated with naturally occurring organic compounds. So far there is no conclusive evidence for the existence of isolated natural organosilicon compounds.1 However, there is an estimation that approx- imately 1010 tonnes of silica are involved in the biogeochemical cycle." Silicon is one of the major components of natural waters (Table 2) occurring in this medium in the form of monosilicic acid.21 The large-scale production and application of silicone compounds seems to have had little ecological impact, even with regard to their stability and environmental persistence. Model studies show that silicones are not biodegradable but can be subject to chemical degradation in the environment. Under the catalytic activity of soil components and its moisture content, silicones can undergo rearrangements and hydrolysis to lower polymerized linear and cyclic products. Higher volatile components can undergo photolytic oxidation in the atmosphere to give water-soluble silanols.Finally, long-term exposure to soil components and sunlight will lead to their ultimate degradation products: carbon dioxide, water and silicic acid.22 Although silicones have liposoluble structures, they do not bioconcentrate in the aquatic environment.' The molecules are not small enough to pass through cell membranes. In contrast, smaller fractions are assumed to be too volatile to endure in aquatic media. There is a suggestion that in vivo, ingested siloxanes undergo simple chemical reaction rather than enzymatically catalysed hydrolysis to silanols (Fig. 2)." In this form, originally liposoluble silicone compounds could be excreted via the kidneys. Although silicon is recognized as an essential trace element participating in the normal metabolism of higher animals, its biochemical mode of action on the molecular level is not very clear.It has been suggested that silicon serves as a cross-linking agent by forming linkages between individual glycoso- aminoglycan chains and joining them to core proteins to form proteoglycans. Glycosoaminoglycans are evolutionary primi- tive, negatively charged unbranched polysaccharide polymers. Proteoglycans themselves are found in all connective tissues and epithelial cells. In this way, silicon deficiency especially affects bone and cartilage formation.23 Silicon is also apparently a major component of osteogenic cells and is present in especially high concentrations in the metabolically active state of the cell, reaching relatively high levels in the mitochondria of these cells.It has been shown that silicon participates in the biochemistry of the subcellular enzyme-containing structures affecting enzymic activity. Interrelationships between silicon and other elements such as molybdenum and aluminium have been established. Silicon-molybdenum contents in plasma and tissues are inversely affected by their mutual presence. A relationship between silicon and aluminium levels in tissues is considered to have relevance to Alzheimer's disease through the formation of aluminosilicate ~lates.~4 It has been postulated that silicon provides strength and elasticity to the connective tissue and biological structures on the basis of the association of decreased elasticity (of the arterial walls in atherosclerosis) to the decreased silicon content (of the arterial walls) through ageing.25.26 There is an estimation that man assimilates 9-14 mg of silicon daily.27 Important amounts of silicon are incorporated in foods such as vegetables, grain, rice and dairy products and in beverages such as beer.28~29 A high-fibre diet usually results in an increase in the intake of ingested silicon.A large fraction of ingested silicon, absorbed across the intestinal wall, is rapidly excreted in the urine. The absorption mechanism of silicon from food, beverages and drinking water and the proportion of absorbed silicon which is retained in the body are not kn0wn.2~>30 Silicone compounds themselves are present in food through antifoaming agents added to canned fruit and vegetable products.Substantial amounts of silicones can be incorporated in alimentary products by their contact with silicone-containing compounds during storage and packaging. It is probable that silicon compounds ingested and absorbed from different sources are submitted to biotransformation, contributing to the total level of organosilicon species in the human body. Literature reports concerning the metabolism of organosili- cons are disperse and mainly refer to pharmaceutical products. R R R R I I I I I I I I OH7R - Si - 0-Si - R __t R -Si -OH + R-Si -0- R R R R Table 2 Concentrations of silicon in some natural waters2' Water Concentration/ mg 1-1 Soil solution 1-40 Streams 0.8-15 Groundwater 3.5-28 Sea- water: Bulk 1-7 Surface 0.0001-0.2 R R R R I I I H I I H++R- Si - 0- Si - R - R -Si - 0 - Si - R I 1 + 1 R R R R R R R R H20+R- Si - 0 - Si - R - R - Si -OH + RSi - OH +H+ R R R R I H I I I I I I + I Fig.2 Possible metabolic breakdown of silicones.'56R Analyst, June 1996, Vol. 121 Many synthesized pharmacological organosilicon compounds are so lipophilic that the excretion through kidneys is negligible. Their biotransformation into more hydrophilic derivatives is mainly supposed to take place in the liver.10 Silicon is introduced in the pharmaceutical industry by another class of interesting products such as pro-drugs. By the silylation of biologically active molecules of drugs having amino or hydroxyl groups, more lipophilic structures are formed which cross lipophilic membranes readily. In vivo, silylated drugs undergo non-enzymic hydrolysis under mild physiological conditions in the body fluids (owing to their chemical lability) or possible enzymically catalysed hydrolysis, releasing the parent drug.10.11 The metabolism and biotransformation of silicones have not been studied in detail.There are no experimental data on the studies of pathways for possible enzymic degradation of silicones under physiological conditions. The nature of inter- actions of silicone compounds with large biological molecules, such as plasma proteins, is another area where there has been limited theoretical and experimental work published. It has been reported that plasma proteins sequentially adsorb to hydrophobic silicone materials and undergo progressive conformational rearrangements leading to their denaturation.Among proteins which most rapidly adsorb are albumin, fibrinogen and immunoglobulin G.31.32 There is also an important theoretical issue concerning the possible immu- nogenicity of silicones. Silicone compounds possess the potential for creating antigen-antibody binding sites. Silicone surfaces provide a major electronegative static charge and electrostatic forces are known to be important for immuno- chemical binding. Moreover, hydrogen bonding (through side groups of silicones) and hydrophobic interactions will also be important in antigen-antibody binding. It has been proposed that the antigenicity of silicone may be in its adjuvant function by inducing conformational changes in native proteins.33-37 Implanted medical devices such as silicone breast implants represent a major potential source of silicone compounds in the human body.Silicone fluid can diffuse ('bleed') out of the envelope and PDMS possibly could migrate into the body. Because of their high relative molecular mass, the diffusion of higher viscosity silicones is not expected. Lower relative molecular mass siloxanes may be small enough for diffusion, each molecular structure having a different potential for biological mobility and activity. It seems probable that some of those compounds could be solubilized in some lipid-containing fractions of body fluids and tissues, according to the conclu- sions of some studies.3*,39 Possible transformations of PDMS, such as hydrolysis, could lead to the formation of monomeric hydroxylated molecules, following pathways similar to those described in Fig.2. General Approach to the Determination of Trace Amounts of Silicones in Biological Materials Although there is an extensive literature dealing with the determination of silicones and the analytical chemistry of silicones in general, there is only a limited amount of work published regarding the presence of silicones or/and their degradation products in biological samples or related matrices. Two possible reasons lie behind this observation. As can be gleaned from the facts cited above, the biochemistry of silicones, biotransformation pathways and metabolism of organosilicon compounds and silicon in general are still very unclear. Accordingly, the particular analytical targets are ill- defined.Second, the determination of silicones, especially in trace amounts, is subject to numerous operating difficulties, mainly connected with their ubiquitous presence and the instability of some species. The determination of silicones in biological materials can be classified as trace analysis, assum- ing that the species measured are fairly uniformly distributed in a matrix, or as microanalysis when the silicone particles are dispersed in a heterogeneous matrix.40 Accidental contamination by omnipresent silicone com- pounds can occur during all steps of the analytical procedure. Stopcock grease, lubricants in syringes, pump fluids, fluids for glassware treatment, silicone rubber for different tubing, sealing, septa or O-ring elements and self-adhering labels are only some of the potential sources of silicones in laboratories.In gas chromatographic analysis, for example, leakage of silicone chromatographic columns at temperatures above 200 "C takes place. Further, it is essential to avoid contamination during sampling procedures. When collecting biological samples, some commonly used items such as talc-powdered gloves or silicone-lubricated syringes should be absolutely avoided. Silicon-specific analyses such as those performed by all atomic spectrometric techniques are especially subject to contamination originating from silicon-containing dust. This makes de-dusting and vacuum cleaning of laboratory space a mandatory component of the trace elemental analysis proce- dure. It is desirable that at least sample preparation should be done under Class 10 or 100 (clean room) laboratory conditions or in cabinets under laminar air How frustrating elemental contamination of all equipment components in the case of silicon can be is very well illustrated for the case in which the concentrations of 18 different elements in plasma were measured in parallel after storage of plasma samples in washed and unwashed plasticware.It was found that only in the case of silicon detection was the washing of plasticware for trace elemental analysis (inductively coupled plasma atomic emission spectrometry) e~sential.~* Some cleaning procedures for the preparation of ' silicone-decontaminated' laboratory plasticware have been described.29 Although generally perfor- med with sound analytical chemical procedures, it is essential in the case of silicone determinations to perform in parallel as many control analyses as possible and also recovery studies simulating the same conditions as for actual samples.The purity of available reagents, solvents and standards is another major concern in silicone determinations. It is necessary to check if the purity grade of all applied chemicals is satisfactory before any actual measurements are performed. Some silicone compounds are unstable, so working solutions should be prepared on a daily basis and stored properly, sealed and kept in a dry place. Another problem is the availability of standard reference materials for biological matrices. In most instances, specific silicone compounds are determined using laboratory standards.Finally, the detection limit is defined as twice the standard deviation of the analytical blank.29.40 Although many of the sources of both contamination and losses of analysed species are observed generally in trace analysis, there are a number of problems specifically associated with analyses for silicones. Silicones are considered to be very stable materials, but their trace amounts are subject to losses of different origin. Molecules such as silanols are especially sensitive and unstable, undergoing condensation even under mild conditions, so that their determination becomes a partic- ularly difficult analytical task. The low heat of vaporization and subsequent rapid evaporation of low molecular mass siloxanes contribute to their losses or sometimes even to cross-contamina- tion between analysed samples.Silicones have a strong affinity for adsorption on glass and all siliceous materials. They also absorb atmospheric m~isture.~O The possibility that a detected species can, in reality, be a rearrangement or reaction product resulting from thermal or catalytic rearrangements of silicones rather than original compounds should always be assumed. Rearrangements of silicones to lower molecular mass species take place in the presence of strong acids or bases.40 If a sample contains traceAnalyst, June 1996, Vol. 121 57R amounts of components having (CH&Si moieties, a hydrolytic cleavage of the Si-CH3 bond can occur, especially in the presence of certain metals (e.g., Fe, A1 and Ca).43 In biological materials, silicones or/and their biotransforma- tion products are usually present in very small (trace) amounts and together with various amounts of other components of physiological origin.Extraction or matrix removal and pre- concentration steps are, therefore, a mandatory component of analytical procedures, which at the same time should preserve the identity and integrity of the original compound. Conse- quently, analytical methods applied to the detection and quantification of species, and ultimately the identification of their specific molecular structures, should be based on a reliable separation procedure and a highly selective and sensitive detection device (analytical instrumentation). This review will discuss some of the basic analytical techniques that have been employed in the area.Survey of Methods Used for Non-specific and Silicon-specific Microstructural Determination of Silicones in Tissues (Microanalysis) Silicone prosthetic devices have widespread application. Al- though often characterized by their supposed biological inert- ness, side reactions and pathological conditions provoked by the presence of silicone take place. This makes experimental techniques for the demonstration of their possible dispersion in the tissue surrounding a particular prosthesis indispensable. The most frequent problem related to the application of gel- filled silicone implants is the potential rupture of the silicone shell elastomer. Several techniques are applied in medical diagnostics for the evaluation and detection of implant ruptures.Deterioration of breast implant capsules is most commonly revealed by film-screen X-ray techniques such as mammo- graphy and ~eromamrnography.~~6 Computed tomography has been reported as a more specific and sensitive technique for the detection of implant ruptures, but patients are also exposed to ionizing radiation.47 Of all techniques used for the evaluation of silicone prosthesis in vivo, magnetic resonance (MR) imaging constitutes the most recent technique and, together with sonography, represents the only non-invasive imaging m0daiity.~5.~8.49 It was reported that MR imaging involving a silicone-selective pulse sequence is highly effective for detect- ing the leakage of silicones from implants.48 Microscopic methods are most frequently used in medical research laboratories for the positive identification of silicone in tissue surrounding the implant and in tissue more distant from the device.Light microscopic examination of tissues is usually applied for the recognition of silicone fragments of optically detectable size range in histological and cytological specimens. There are difficulties in visualizing silicone in tissues using conventional light microscopy because generally silicone is refractile, non-polarizable and non-stainable. Alternative light microscopic techniques such as phase-contrast and dark-field microscopy have been proposed to improve silicone detec- tion.50 More detailed studies usually use light microscopy only as a screening device for the determination of silicone-containing tissue areas. Conclusive identification of silicon components is subsequently performed by electron microscopy coupled with X-ray analysis.In electron-probe X-ray spectrometry (EPXRS), analytical microcharacterization of a tissue sample is obtained by focusing an electron beam of moderate energy on to the microscopic area of the sample at the location where elemental composition is to be determined. The atoms in a very small volume of the sample are excited by the incident electrons and, on returning to the ground state, emit X-rays characteristic of the excited elements. In SEM, the sample image is obtained by either backscattered or transmitted electrons. The X-rays produced in SEM are usually detected and measured by applying energy-dispersive X-ray analysis (EDXA) for quanti- tative elemental analysis.Electron probe microanalysis has been applied (to cite only some of the numerous studies) to identify and demonstrate the extent of silicon-containing material within tissue blocks.51-57 Identification of the silicon spectral peak in an X-ray energy spectrum in these studies was correct, but information regarding the chemical form of the silicon-containing compounds could not be provided. Elec- tronic transitions which are obtained from X-ray frequencies applied during X-ray analysis are independent of the type of bonding of silicon with other atoms and therefore cannot be used to elucidate the chemical structure of compounds. Silicon-specific (Elemental) Techniques for the Detection of Silicones in Biological Materials Atomic spectrometric techniques are by far the most frequently applied methods for the determination of total silicon in a variety of biological materials.Unfortunately, the information obtained from these methods is limited because they are incapable of revealing the structure of original silicon-contain- ing compound. Electrothermal atomization (sometimes known as graphite furnace) atomic absorption spectrometry (ETAAS), is generally the most frequently used technique in laboratories for the determination of traces of silicon in biological samples. Compared with flame atomic absorption spectrometry (FAAS), its detection limits are 10-100 times better, and analyses are performed with microlitre levels of solubilized sample.58 Inductively coupled plasma atomic emission spectrometry (ICP AES) is also a very sensitive technique providing high temperatures and an inert atmosphere of the discharge.Together with direct current plasma atomic emission spectroscopy (DC AES), it presents a technique which is also widely applied in trace elemental analysis laboratories. When ETAAS is applied, a necessary step during the preparation of samples is the addition of chemical modifiers that retain the analytes at higher temperatures in the furnace and aIlow volatilization of interfering matrix compounds. It is frequently the case that lower relative molecular mass silicones are inclined to volatili- zation and disappearance from the system before atomization and actual measurement. Addition of calcium compounds is based on an enhancing effect of calcium on the absorbance of silicon.29.59 Introduction of a graphite furnace oxidation step to eliminate organic interferences is also rec0rnmended.~9 It is important to mention that earlier studies involving spectrophotometric methods were based on the formation of silicomolybdate (Si02.12M003) for the determination of sili- con in biological fluids and tissues.51.6662 The major analytical concern in all these studies is the elimination of phosphorus, which is usually present in large amounts in biological samples and interferes with the spectrophotometric determination of silicon.A number of atomic spectrometric methods have been developed for the measurement of the total silicon content in blood and other body fluids such as urine.59,63-68 Many studies involved measurements in the body fluids of patients with renal failure. The results indicate a decrease of silicon excretion through urine and an increase (accumulation) in other body fluids, mainly blood, compared with levels considered to be ‘nomal’.28769-80 Higher silicon contents were also measured in tissues of patients with renal failure.81 A study also reported traces of silicon in tissues of patients submitted to haemodi- alysis, where the origin of the silicone was attributed to tubing incorporated in the dialysis apparatus.82 Somewhat elevated concentrations of elemental silicon were measured in the blood5 8R Analyst, June 1996, Vol.121 of patients with silicone breast implants.29.83.84 ETAAS con- firmed the presence of silicon in tissues of patients with silicone breast prosthesess5 via an already developed method for measuring silicon in heptane extracts.86 Higher silicon levels were also measured in tissues of cadaveric patients with silicone implants, when nitric acid digestion and subsequent DC AES measurement were employed.87 It is important to take into consideration that methods involving extraction of silicon-containing compounds with organic solvents and subsequent elemental analysis for total silicon content of extracts give only partial and very vague information with respect to silicon speciation.Organic solvents can extract liposoluble silicone compounds but also possibly some inorganic silicon species in colloidal form. Also, there is great uncertainty regarding the possible nature of association with proteins and the subsequent solubility of these species in organic solvents.From the analytical point of view, it is very interesting to compare different results for 'normal' silicon levels in blood and its components obtained in different studies (Table 3). The methods applying FAAS obviously lack sensitivity. Some extremely high measured levels indicate severe contamination, which are inevitable if all the previously discussed precautions are not considered as part of the analytical procedure. Possible accidental contamination is also frequent and can be traced in most of the cited studies by high silicon concentrations in some controls in comparison with lower levels in patients where higher concentrations are actually expected. These variations can also be caused by silicon ingestion and the level becomes elevated through sources such as a high-fibre diet or higher silicon levels in drinking water.28 Generally, the values in Table 3 indicate that silicon-containing species are likely to be found in plasma.A good statistical analysis is necessary for accurate and reliable presentation of results in these types of study. Some studies involving the determination of elemental silicon in foods are potentailly interesting in relation to biological matrices. Silicone (PDMS) defoamers were extracted from fruit juices and beer with chloroform and measured by FAAS.88 A collaborative study reported good results with PDMS extraction with 4-methylpentan-2-one from pineapple juice and measurements with FAAS.89 By acidifying fruit juice samples with hydrochloric acid before extraction, better phase separation was obtained.g0 Silicones, added to edible fats and oils as defoamers and antioxidants, could be measured directly by ETAAS after dilution with isooctane, applying furnace air oxidation for reduction of matrix interferences.91 Survey of Silicone-specific Techniques IR, FTIR and Raman Spectrometry Silicones can be very easily identified by their IR spectra because of the high specificity of the spectral patterns of organic groups attached to silicon, although differences in relative molecular mass or relative molecular mass distribution could be overlooked.In contrast, UV spectrometry is not a very good tool for identifying silicones because of their lack of absorption of UV radiation, except for aromatic substituents.IR spectrometry is especially useful for the identification of silicone molecular structures and for quantitative analysis when well characterized reference standards are available. The strong absorption of the siloxane part of a silicone molecule makes it a very distinctive feature in an IR spectrum. As expected, IR spectrometry is not sensitive enough for trace analysis. Better sensitivity for trace analysis can be obtained by preconcentration with solvent extractions or applying prior gas or liquid chromatographic separation. In trace analysis, the most characteristic band is that originating from the symmetrical methyl deformation [(CH3)2Si] at 1262 cm-1 (wavenumber). This is used as an isolated band for quantification with minimum interferences from the matrix.92.93 Raman spectrometry is a very attractive technique for the characterization of biological materials because it can be performed on neat samples.The same applies to attenuated total reflectance (ATR) IR spectrometry of biological samples. This technique permits the study of the surface of a sample when it is too thick for transmission methods. The penetration depth of the IR beam into the sample is a function of wavelength, angle of incidence of the IR beam and the relative refractive index and is usually expected to be several micrometres. For the analysis of solid microsamples, FTIR spectrometry is a preferred technique because of its energy throughput advantage over dispersive IR techniques.92 An early study reported the use of IR spectrometry for the determination of trace amounts of silicones in foods and biological samples such as lung tissue, blood and animal organs.94 Silicones could be extracted from a matrix with benzene or chloroform if direct extraction in carbon disulfide is not possible.It was reported that the final concentration of silicone in the solvent should be at least 50 ppm (0.05 mg ml-1) for measurements in a microcell. Basically the same method was applied for the detection of silicone defoamers in beer and yeast samples, but low recoveries were obtained and also the spectra exhibited many peaks due to interfering species. This Table 3 Normal total silicon levels in human blood measured in some studies Ref. King et a1.60 Lo and Christian63 Mauras et al.69 Dobbie and Smithw Berlyne and Carus065 Matusiewicz and Barnes66 Roberts and Williams7' Gitelman and Alderman79 Hosokawa and Y o ~ h i d a ~ ~ Marco-Franco et al.76 Bercowy et a1.68 Malata et al.83 Peters et al.29 Teuber et al.84 Year Technique 1955 Spectrophotometry 1978 ETAAS 1980 1982 1983 1984 1990 1990 1990 1991 1994 ICPAES FAAS ETAAS ICPAES DCPAES ETAAS ETAAS ICPAES DCPAES 1994 ETAAS 1995 ETAAS 1995 ICPAES Blood component Serum Whole blood Serum Whole blood Serum Serum Serum Plasma Plasma Serum Serum Whole blood Plasma Serum Whole blood Whole blood Serum Concentration/ vg 1-' (ppb) 1400 1490 770 110 600 f 125 309.6 f 82.1 140 f 14 170 f 100 220 f 80 10-250 < 300-5000 < 400-4000 < 200-4000 42-366 130 k 70 2600 24.23-26.66Analyst, June 1996, Vol.121 59R was supposed to be overcome by the application of acetone- benzene as extraction solvents and NaOH for the removal of interfering components. The recoveries were improved but the introduction of the base probably contributed to the partial cleavage of siloxane b0nds.~5 Some early analytical studies performed on human tissues reported the application of the IR technique for the positive determination of silicone structures. Silicones were detected in brain and kidney tissues of patients submitted to heart-lung machines, where silicone defoamers are added to the bubble oxygenator.96 After extraction with benzene and purification of extracts on alumina columns, silicones dissolved in dichlo- romethane were confirmed by IR spectrometry. In another study silicone particles, located by light microscopy in capsular tissues of silicone breast implants patients, were confirmed by means of the same te~hnique.9~ A laser Raman microprobe was used to detect silicones in the lymph node of a patient with a silicone elastomer finger-joint prosthesis.98 Small sections (2-20 pm) of the specimens were mounted on sapphire sample supports and irradiated with 5 14.5 nm green radiation from an argon-krypton laser for the collection of the Raman scattered radiation.The main limitation of the method is in its sensitivity, which mainly depends on the Raman scattering efficiency of the analyte compound in a given matrix, and the distribution of the Raman scatter (analyte) by the matrix. Studies were performed using FTIR-ATR analysis to evaluate in vivo adsorption of blood components (fibrin, cellular elements) on silicone rubber99 or on silicone elastomer membranes by exposure to flowing blood.100 The same technique was applied to evaluate protein adsorption on silanized surfaces of polymethacrylic soft contact lenses.101 A method was developed for the detection of silicones (2-12 mg per gram of tissue) in a rabbit muscle tissue adjacent to silicone implants, based on FTIR-ATR analysis of methylene chloride extracts.Some recent studies extensively used FTIR microspec- trometry for the detection of silicone structures in breast tissues of silicone implant patients.103-105 Usually, a removed implant is applied as a control 'fingerprint' for spectral confirmation. In some instances the FTIR spectra of tissue samples revealed the presence only of an Si-0 stretch without an Si-C stretch which is present in original silicone gel.'W This suggests a possible hydrolytic cleavage of the silicon-carbon bond of silicone.Near-IR Raman spectrometry with laser excitation at 782 nm (reduced fluorescence interference compared with excitation at 5 14.5 nm98) was also used for the detection of silicone in lymph nodes of breast implant patients.106 It was suggested that the method could be extended to the in vivo analysis of living tissues. NMR Spectrometry Silicones are mainly composed of atomic nuclei which resonate in a magnetic field, which makes them responsive to the NMR technique. Most of the functional groups attached to silicon have distinctive proton resonances so that lH NMR is generally used for qualitative and quantitative analyses of silicones.However, 29Si NMR has become widespread in recent times and is the technique of choice for structural differentiation, because of its ability to distinguish between different silicone species and the specificity of its response.107 Quantitative NMR spectra are usually applied for the determination of structure and the analysis of mixtures. NMR is generally not used in trace analysis because of its lower sensitivity compared with other techniques. However, the information obtained by Garrido and co-workers108-112 on the migration and biodegradation of silicone compounds in differ- ent tissues and blood samples of model animals and silicone implant patients, by means of NMR, seems to exceed the results of all other studies performed so far. In the course of the cited studies, 1H NMR spectrometry was used for the examination of the degradation of silicones in vivo and 2% NMR spectrometry, with a broad-band magic angle spinning (MAS) probe (to eliminate broadening from dipole- dipole coupling and chemical shift anisotropy), was employed for the analysis of tissue and blood samples in vitru.Migration and biodegradation of silicones in breast implant patients were reported.ll1 The presence of unchanged silicone (peak at -22 ppm), hydrolysed silicone where methyl groups are substituted by hydroxyl groups (peaks at -30 to -80 ppm), silica (peaks at -90 to - 1 15 pprn), pentacoordinated silicon complexes (peaks at -120 to -150 ppm) and alkyl- and hydroxyl-terminated silicone (peaks at 0 to -15 ppm) were detected. The concentrations of silicone in examined blood samples, accord- ing to this study, exceed by 1000-10000 times the average concentrations of elemental silicon obtained in similar studies where AAS was used.29,83,84,113 The application of lH NMR spectrometry in a localized manner for the detection of silicones in the liver of breast implant patients in vivo resulted in a sensitivity to ppm levels of silicon.112 Gas Chromatography and Gas Chromatography-Mass Spectrometry Methods Gas chromatography and GC-MS are appropriate for the molecular identification and structure determination of sili- cones and for the sensitive determination of trace amounts of these species in biological materials. Separation (using high- resolution capillary columns) together with identification on the basis of retention times and quantification of silicones by GC, usually with flame ionization detection (FID), is a common procedure.In contrast, LC is rarely used in analyses for silicones because of their non-polar nature, which makes them unsuitable for LC separation. The qualitative capability of the MS detector improves the performance of GC, making the GC- MS method the most sensitive and selective technique for the quantification of specific silicone species. The nature of silicone compounds and their fragmentation patterns impose many restrictions on their determination by GC-MS. The high boiling point of high molecular mass PDMS renders them unsuitable for GC separation. Some unstable silicone species such as silanols must be derivatized before introduction into the GC column in order to protect them from condensation.The unambiguous identification of both larger linear (> MDSM) and cyclic ( > D6) siloxanes through their electron impact (EI) mass spectra is generally not possible, because of the predominance of identical rearrangement ions regardless of the original structure. It is recommended that the best clue for the characterization of silicones is through a combination of the structural information given by EI fragmen- tation patterns and the molecular mass information provided by the chemical ionization method.' l 4 One of the important problems in GC-MS analysis is that GC-MS systems are major sources of background silicone through PDMS stationary phases which are generally applied for GC separation and also through components such as septa and seals made of silicone rubber or silicone diffusion pump fluids.Among the numerous studies dealing with both the GC and MS or GC-MS of silicones, only a small number were concerned with the presence of silicones in biological materials or related matrices. 2,6-cis-Diphenylhexamethylcyclo- tetrasiloxane (Fig. 3), a drug proposed for the treatment of prostate carcinoma, was determined in human serum with a reported sensitivity of 0.1 ng ml-1 by means of mass fragmentography after extraction with heptane.115 The60R Analyst, June 1996, Vol. 121 hydroxylated products of the same drug [(a) dimethylsilanediol, (b) methylphenylsilanediol, (c) trimethylphenylsiloxanediol and (4 phenol (Fig.3)] obtained by biodegradation were determined in urinary samples at ppm levels by means of GC- MS. l6 The derivatization of hydroxylated metabolites ex- tracted with diethyl ether-ethanol was performed using hexam- ethyldisilazane (HMDS) in an acidified medium. HMDS, in the presence of catalytic amounts of acetic acid, as a mild reagent does not promote the formation of new siloxane bonds. It was reported that the silyl derivatives obtained have suitable GC properties with respect to their stability and volatility. Although satisfactory results were obtained after the evaporation of samples and extracts containing hydroxylated silicones, it is possible that both the evaporation and derivatization processes can result in partial or even total loss of the species under study due to their degradability, reactivity and volatility and also their expected low concentrations.Gas chromatography and GC-MS methods for the determi- nation of water-borne organosilanols and water-insoluble organosilicons have been developed with the possibility of detection of ppm to ppb levels of silic0nes.~3 The derivatization of silanols with hexamethyldisiloxane (HMDSO) was per- formed after digestion with hydrochloric acid. Further, HMDSO is employed as a selective, in situ, trimethylsilylation and extraction agent. On the basis of the previous method, a procedure was developed in which PDMS oligomers were determined in connective tissue around mammary prostheses at a detection limit of 6 pg g-1.117 Unlike other methods, where laboratory standards of chemical species under investigation were used for quantification, the GC quantification was performed using octamethyltrisiloxane and the silicone levels were expressed as dimethylsiloxane equivalents. The same method was applied for the evaluation of silicone levels in human milk.118 The application of the method demands extreme caution in order to eliminate possible losses of unstable silicone compounds.Also, the demand for silica-free and acid-resistant equipment, such as tightly sealed high-quality PTFE plastic- ware, makes this procedure extremely expensive for routine analysis involving numerous samples. The method is not suitable for phenyl-substituted silicones because of possible acid-catalysed hydrolytic cleavage of phenyl-silicon bonds.It was reported in an early study that silicones could be extracted from the blood of breast implant patients and determined by MS, but no details on the analytical procedure applied were given. 119 Experiments involving measurements of linear (MM- MD13M) and cyclic (D3-D9) silicones in fish tissues at a detection limit of 0.3 pg g-1 using GC and GC-MS, after the Ph Me I I .Si - 0 -Si I Me I Me I :H3 Ph I Ph 743 HO- Si - OH HO- Si - OH HO- Si - 0 - Si - OH D O H I I I I CH3 CH3 CH3 CH, ( 4 ( b) (4 (4 Fig. 3 Structure of 2,6-cis-diphenylhexamethylcyclotetrasiloxane and its biodegradation products: (a) dimethylsilanediol, (h) methylphenyl- silanediol, (c) trimethylphenylsiloxanediol and (6) phenol.* addition of silicones to the food, showed that only small amounts of these compounds were retained by the fish after 6 weeks of feeding, in contrast to much higher absorption of PCBs. l*O Silicones were extracted from tissues with hexane. GC-MS was used for the confirmation of particular silicone structures and capillary GC with FID was applied for determi- nation of trace levels of silicone. Evaporation of lower relative molecular mass species took place during the preparation of samples. Some other studies involving GC-MS methods for the determination of silicones in the environment could potentially be extremely valuable for the detection of trace amounts of silicones in biological matrices. A sensitivity of 0.2 ng ml-1 was achieved by the purge-and-trap GC-MS method described for the determination of octamethylcyclotetrasiloxane (D4) in water.I21 Since the method is extremely sensitive it could suffer from background contamination coming from other volatile materials present in the laboratory environment, such as solvents.PDMS associated with airborne particles were collected with Teflon filters and extracted with 1,2,2-trifluoro- 1,l72-trichloro- ethane (Freon 113) or dichloromethane.122 It was reported that silicone fluid at a detection limit of 0.1 ng was determined by pyrolysis GC-MS, by applying a correlation between major identified pyrolysis products (cyclic siloxanes) and the starting silicone material. One of the major problems related to the application of the cited methods to real biological samples (the described work was performed mostly on synthetic samples) is connected with analytical standards.Uncertainty regarding the presence of actual compounds in the samples under investigation and the unavailability of actual silicone compounds render the quantita- tive detection of silicone biodegradation products a difficult problem with an extremely questionable outcome. The demand for structurally close compounds as internal standards some- times conflicts with features of other steps in the applied procedure, making the choice of a suitable internal standard another problem for the analytical chemist (e.g., ions of different m/z values must be produced for mass spectra). Although GC-MS seems to be an ideal technique for the identification of silicones and their biodegradation products in biological samples, the difficulties described above substan- tially reduce the number of reported and successful relevant studies. Problem of Speciation of Silicon and Identification of Silicone Biotransformation Products in Biological Samples One of the most important considerations in the many analytical studies discussed above is the identification of small molecules or biomolecules incorporating biological silicon or silicone biodegradation products, Some of the cited ~tudies,1~~109-~ 1 where positive identification of biochemically modified sili- cone species was reported, are not persuasive in terms of the methodological approach and, accordingly, the positive con- firmation of the conclusions derived from experimental results cannot be accepted.Under physiological conditions, the possible existence of biodegradation products associated with siloxane bond cleavage leading to hydroxylated silicone compounds116 seems a more probable mechanism for bio- transformation of silicone compounds than the cleavage of silicon-carbon bonds with the same O U ~ C O ~ ~ . ~ ~ ~ , ~ ~ ~ Inter- actions of silicones with proteins is another issue with more theoretical assumptions than experimental confirmation. If the existence of silanol degradation products is assumed, then their association with proteins via hydrogen bonding is very likely. The formation of associations where components of protein functional groups are pentacoordinately bonded to the silicon atom could be another alternati~e.*~3 It was suggested that61R Analyst, June 1996, Vol.121 Table 4 Techniques for the analysis of silicones in biological matrices Technique Application X-ray methods Atomic spectrometric methods Elemental microcharacterization of tissue samples Elemental determination of silicon in biological materials FAAS ETAAS, ICPAES and DCPAES IR spectrometry FTIR and Raman spectrometry NMR spectrometry GC and GC-MS Determination of silicones in extracts from biological materials Determination of silicones in solid biological microsamples Determination of silicones in biological samples in vivo and Determination of silicones in extracts from biological materials in vitro Level of sensitivity mg g-' mg 1-' 1-' mg 1-l mg g-' Pg 1-' mg 1-' silicon is only unspecifically adsorbed to serum proteins.lz4 In this study, HPLC was applied for separation and ETAAS for the detection of silicon-containing fractions.Gel electrophoresis is another separation technique that could be applied to give information on particular protein binding. Some other techniques which are still under development, such as matrix-assisted laser desorption/ionization (MALDI) MS and electrospray MS, which permit the measurement of intact molecular ions of high molecular mass biopolymers, could yield valuable information on silicone and silicon binding to proteins in future work. Conclusions Table 4 summarizes analytical techniques used for the determi- nation of silicones in biological samples and described in this review. Although most of them are distinguished by relatively high reported sensitivities and capabilities to examine different kinds of biological samples, only the methods based on atomic spectrometric techniques have been widely used for the determination of silicone levels in biological samples.It is evident that although an important interest in the determination of silicones in biological matrices exists, the identification of silicone biodegradation products is the most significant problem which has not been solved so far. 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