Anal. Chem. 2009, 81, 4679–4694
Pharmaceuticals and Related Drugs R. K. Gilpin* Brehm Research Laboratory University Park, Wright State University, Fairborn, Ohio 45324-2031 C. S. Gilpin Selectosep, LLC, 111 West Main Street, Freeport, Ohio 43973 Review Contents General Separation-Based Methodology Spectrometric-Based and Other Methodology Literature Cited
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The current article surveys pharmaceutical and related methodology that has appeared in the literature between January 1, 2007 and December 31, 2008 and is directed exclusively toward the analysis of compounds in either their bulk or formulated forms. Emphasis is placed on procedures for evaluating their quality, purity, strength, and stability. The review does not deal with biochemical, clinical, metabolic, pharmacokinetic, or related aspects of the topic nor does it include the analysis of pharmaceuticals in the environment or food products. Because of space limitations, the cited articles represent only a small fraction of the total number of those published, and even though a cited paper may deal with more than one technique, it is discussed typically once in the paper. In the selection of appropriate citations, an attempt was made to place greater emphasis on procedures related to newer compounds, emerging and/or highly used techniques, unusual approaches and methodology, and more comprehensive studies with only a few examples cited from the very large number of publications that discuss either more routine procedures, less often used techniques and approaches, or older compounds. The current review is organized similar to the last review in this series that appeared 2 years ago in Analytical Chemistry (1) except the general section has been expanded significantly and the techniques section reduced slightly. This was done to place more emphasis on general trends and emerging techniques and less on more routine methodology. Thus, the article is organized into three major sections: General, Separation-Based Methodology, Spectrometric-Based and Other Methodology. These sections are arranged in terms of specific approaches and applications. GENERAL Although books and book chapters are not included in the current review, as has been the case for many years, the reader is referred to an article that recently appeared, which is based on books and major monographs published (2). It discusses the changing nature of pharmaceutical analysis over a 25 year period with an emphasis on separation methodology. The article also contains a section on regulatory issues that stresses the impor* To whom correspondence should be addressed. 10.1021/ac900804d CCC: $40.75 2009 American Chemical Society Published on Web 05/06/2009
tance of the “globalization of the drug market” and its influence on the changing nature of safety and efficacy and how these affect analytical requirements. The author of the above review also notes that caution should be exercised in “believing that possession of up-to-date, automated/computerized (and very expensive) instruments and validated methods automatically give good and reliable results.” Likewise, these feelings are expressed by the authors of the current review with the additional observation that many manufacturers and purchasers of modern instrumentation believe smaller and more automated equipment is better. Clearly from a space and time management point-of-view this may be true, but this type of instrumentation often is less useful for solving more demanding and nontraditional types of analytical problems. The exceptions to this trend are in the fields of nuclear magnetic resonance (NMR) and ion cyclotron resonance (i.e., Fourier transform mass spectrometry) spectroscopes, where the field strength of spectrometers continues to increase. Over the last 2 years, many articles were published that are more general in terms of analytical methodologies that consider either specific compounds, types of problems, or developments in instrumentation. A few examples of articles that deal with a more comprehensive overview of analytical methodology associated with particular compounds or classes of compounds are ones that discuss cisplatin (3), cephalosporin antibiotics (4), furosemide (5), morphine (6), omeprazole (7), pegfilgrastim (8), sertraline (9), ribavirin (10), thalidomide (11), and water-soluble vitamins (12). Besides these general analytical profiles, the results from other studies address more specific issues related to such topics as the affect of powder pH on the solid state properties of amoxicillin trihydrate (13), the oxidation of Zn2+-insulin by redox active transitions metals (14), and the influence of low levels of inorganic impurities on the important physicochemical properties of paracetamol (15). A variety of techniques were employed in carrying out the latter study, including inductively coupled plasma mass spectrometry (ICPMS), scanning electron microscopy energy dispersive X-ray microanalysis (SEM-EDX), X-ray power diffraction analysis (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and inverse gas chromatography (IGC). Traces of aluminum were found to be present at the 0.1-5.6 ppm level depending on the batch, which, in the presence of other trace inorganic impurities, was found to have a measurable effect on the finished product’s performance. Listed sources of possible contamination were packaging materials, catalysts, electrodes, Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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reagents, and solvents used to synthesize the active ingredient. In addition to the above compound related publications, many other articles and reviews were published that consider a variety of general methodology and compound property topics including ruggedness and robustness (16), multivariate calibration (17), validation of interlaboratory studies (18), a molecular screening database for chiral compounds (19), impurity profile tracking (20), a general strategy for identifying package leachables (21), methods for monitoring the purification of monoclonal antibodies (22), and the measurement of physical properties such as solubility (23), pKa (24, 25) and lipophilicity (26-30). The latter citation is a comprehensive review of modeling as it relates to reversed-phase liquid chromatographic data and quantitative structure-retention relationships (QSRR). Many different molecular descriptors and modeling methodologies are considered, including principle component analysis, decisions trees, artificial neural networks, partial least-squares, stochastic gradient boosting, multivariate adaptive regression splines, etc. In addition to this paper, another discusses new software (i.e., GlycoMiner) for elucidating the composition of glycopeptides using tandem mass spectrometry (MSn) data, as well as date generated using highperformance liquid chromatography-mass spectrometry (HPLC-MS) (31). Reliability of the software was tested using tryptic digests of human R-1-acidic-glycoprotein (AGP) and transferrin, which have five and two N-glycosylation sites, respectively. A total of over 3000 MS/MS spectra were examined with an observed 0.1% rate of false positives and false negatives. On the basis of this work and the increasing interest in protein-based drugs, the program appears to be a useful tool for studying glycosylation, which is one of the more complex and frequent forms of post-translational modification with at least half of mammalian proteins known to undergo this process. Many other general studies have been carried out to examine various aspects of, and to answer questions related to, formulation, drug delivery, and manufacturing. They have dealt with a host of topics including (1) the use of more exotic formulation materials for sustained release and site selective delivery of the active ingredient(s); (2) procedures for monitoring process purification and drug degradation during manufacturing; (3) the influence of drying on the physicochemical properties of the bulk and formulations; (4) the development and characterization of new excipients; and (5) drug-excipient interactions and formulation degradation. One of the many examples of the work that has been carried out in these areas is the potential use of carbon nanotubes as novel drug delivery systems including their synthesis, purification, and analytical procedures to characterize them (32). This is one of several papers that deal with the potential pharmaceutical uses of nanotubes as formulation excipients. Besides nanotubes, in a number of related cases, other types of nanostructures (e.g., nanoparticles and complexes) and biodegradable materials also are being examined as potential drug delivery systems to either administer the active over extended periods or increase its effectiveness by targeting the active drug to site specific locations. A comparative study was carried out to evaluate the effectiveness of polyelectrolyte nanocomplexes and nanoparticles for insulin delivery in order to protect it against enzymatic degradation 4680
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and to enhance its transport across the intestinal mucosa (33). The release of insulin from the trimethylchitosan-based nanostructures and their stability under simulated gastrointestinal conditions were monitored using a combination of high-performance liquid chromatography (HPLC) and dynamic laser light scattering. In a second study, lipid nanopariticles were evaluated for the sustained topical delivery of ketoprofen and naproxen (34), and in a third study, polymeric implants were examined as a means of delivering tamoxifen citrate to the tumor site(s) (35). The end application for the poly(sebacic acid-co-ricinoleic-esteranhydride)-based implants is for the treatment of breast cancer. Again HPLC methodology was used to measure the active drug and differential scanning calorimetry to characterize its solid-state properties. Process related problems continue to be of concern, and many papers have appeared that are related to the development and application of methodology for studying them. One example of these is the use of different drying procedures for the preparation of protein-based pharmaceuticals and how they affect the quality of the final product(s) (36, 37). In these two papers, various properties of the target proteins are considered in terms of specific surface area, composition heterogeneity, secondary structure, and molecular dynamics. Likewise, other papers deal with the detection of lot-to-lot variations in the amorphous microstructure of lyophilized protein formulations (38), the influence of chitosan on the release of proteins from poly(organophosphazene) hydrogels (39), and the glycosylation of therapeutic proteins during storage as formulated infusion products (40). In the latter instance, the degree of degradation was characterized using a combination of a boronate affinity chromatographic method to quantify the glycosylation products in different antibody and infusion formulations and high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) to determine the degree of glycosylation per molecule. Besides the above compound and topical reviews, many general papers and reviews have been published that deal specifically with analytical approaches and associated software/ hardware, especially separation and spectrometric instrumentation. There has been increasing interest in hydrophilic interaction chromatography (HILIC), since its reintroduction in 1990 based on earlier work completed and published in the 1970s. Separations are carried out using hydroorganic eluents that contain high levels of the organic component in combination with a hydrophilic stationary phase. General reviews have been published that deal with the method development aspects of HILIC (41) as well as the influence of column temperature and the choice of eluent components on selectivity (42). Likewise, another publication has appeared that discusses its bioanalysis potential for polar drugs when coupled to mass spectrometry (43), and a fourth how combining it with conventional reversed-phase methodology can broaden the elution window for the analysis of difficult to separate compounds (44). The latter citation is a good source of additional references (i.e., reviews and general papers) that cover various aspects of the technique including its use in proteomics research. Another reemergence of an older technique, which was first discussed in the early 1980s by one of the authors of the current review, is the use of totally aqueous HPLC conditions (45, 46). However, much higher eluent temperatures have been employed
in this more recent work to reduce analyte retention. Several other published accounts of the applications of both hydrophilic interaction and totally aqueous liquid (i.e., “green separations”) chromatography to specific separation problems have appeared over the past 2 years and can be found by searching the scientific literature using the appropriate keywords. The increasing use of hyphenated techniques continues to be an important trend in pharmaceutical methods development and drug characterization, especially high-performance liquid chromatography-mass spectrometry, and to lesser extent capillary electrophoresis-mass spectrometry (CE-MS), high-performance liquid chromatography-nuclear magnetic resonance spectroscopy (HPLC-NMR), and high-resolution gas chromatography–mass spectrometry (HRGC/MS). Two papers have appeared that discuss the use of nanoelectrospray ionization (nESI) in combination with high-field asymmetric waveform ion mobility spectrometry for analyzing biopharmaceuticals (47, 48). Proof-of-concept of the technique and fundamental principles of the ion physics/ migration mechanisms are presented in the cited references. However, in the first paper, many different types of compounds are considered including pharmaceuticals and in the second the paper is targeted toward the use of the technique for the bioanalysis of drugs. A recent review has appeared that discusses the reemergence of quantitative HRGC–MS (i.e., using time-of-flight spectrometers) in terms of both one and two-dimensional separations (49). For the most part, benchtop spectrometers are being used to carry out this type of analysis limiting the mass accuracy of the MS measurements. There are many areas of analysis (i.e., types of compounds) where HRGC combined with high-resolution TOFMS would offer greater advantages in terms of ease, reliability, and sample throughput. Unfortunately, although current highresolution TOF instruments can provide resolutions of 20 000 in the single pass mode and about double this in the double pass mode, many of them are not equipped with appropriate interfaces and ionization sources to allow GC experiments to be carried out with them. One of the factors that appears to have had a significant influence on available GC–MS instrumentation is the general lack of interest in the use of GC methodology in the pharmaceutical industry based on the relatively few publications appearing in the current literature vs the number that appeared 2-3 decades ago. Similarly, because of the large yearly investments in analytical instrumentation by the health care industry, it also has had a profound influence on many other major types of equipment being developed and marketed in terms of availability of certain types of hardware and analytical capability. It would be interesting to track other trends in the development and availability of analytical instrumentation in terms of their use in the pharmaceutical and biomedical fields of research. Combined (i.e., hyphenated) techniques have been useful for investigating a wide range of problems including profiling the composition of natural and herbal products. The uses of these techniques continue to grow as they relate to the quality control of Chinese herbal products (50, 51). Representative examples of some of the findings being published in this area are included later in the current review. Likewise, evaluating the purity and stability of bulk drug substances and formulated products containing them have been other important applications where hyphen-
ated techniques are especially useful. Generalized procedures have been described for evaluating the purity, identity and concentration of large compound files that employ a combination of various HPLC-detection methods including ESI-MS (52). The reported throughput for the approach is 1 min per sample. Other examples of the use of hyphenated techniques to quantify specific pharmaceuticals and/or study their purity and stability are included in their respective subsections below. In terms of hyphenated hardware development, a paper was published recently that describes the coupling of microemulsion electrokinetic chromatography with mass spectrometry by using an atmospheric pressure photoionization (APPI) interface (53). Performance of the approach/hardware compared to more conventional capillary electrophoresis-electrospray ionization-mass spectrometry instrumentation was evaluated using various β-blockers, central stimulants, and diuretics and found to be comparable in terms of performance. In addition, an interface has been fabricated to couple planar chromatography with mass spectrometry (54). It uses an automated extraction-injection procedure followed by conventional HPLC-ESI-MS to perform the analysis in a reported “hands-free” manner. Performance results are given for quantifying caffeine in drinks and pharmaceutical tablets; however, no internal standard was used in making the measurements. As a general trend, as has been the case for over 3 decades, HPLC continues to be unchallenged as the most often employed technique for assaying various pharmaceuticals. The breadth of applications that use some form of HPLC span a very broad range. The bulk of the content and stability assays continue to be carried out under reversed-phase (RP) conditions, and the most common approach is to use some type of standard 150 or 250 mm column packed with 5 µm porous silica-based hydrophobic materials in combination with UV detection. Although many assays are published that employ single wavelength detectors, photodiode array detectors have become both popular and common in many laboratories, since they provide a convenient means of monitoring several wavelengths at once for maximizing sensitivity and assuring peak purity, as well as acquiring “on-the-fly” spectra for complex mixture profiling. In order of usage C18, C8, and CN surfaces are the most common types of reversed-phase packings with a majority of the separations being carried out on standard octadecyl modified silica or based on deactivated octadecyl modified silica. Although standard column technology is the frontline approach for most quality control methods, the number of assays that use either monolithic or smaller particle-based columns continues to increase, especially as it relates to high throughput and biological separations. Many reviews and general papers have appeared that discuss various aspects of column technology. Four of the most common areas of interest are monolith construction (55-57), molecular imprinted polymers (58, 59), alternate nonsilica-based substrates (60, 61), and small particle silica-based materials (62). Besides silica, zirconium and titanium oxide materials are two popular substrates. Likewise, column selection and methods of classification of them continues to be an important topic (63-65). In the latter citation, three different column classification methods (i.e., proposed by other researchers) are compared as they specifically relate to pharmaceutical separations. Analytical Chemistry, Vol. 81, No. 12, June 15, 2009
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In cases where sub-3.0 µm packing are being employed, higher pressure eluent delivery systems are required. Although commercial pumps are available that can be operated in the 1000 bar and greater pressure range, some investigators continue to assemble their own systems, which can be operated at higher pressures. Unfortunately, when ultrahigh pressures are employed, fluid compressibility, which can be and generally is ignored in conventional HPLC, becomes an important consideration. The result of using ultrahigh pressures is nonlinear heating and retention behavior that is different than that expected. Studies of these effects and development of appropriate physicochemical models for them are emerging areas of research interest (66). Various aspects of ultrahigh pressure liquid chromatography (UPLC) (67, 68) and its application in pharmaceutical development (69-72) has been discussed by a number of investigators. Additionally, a paper has appeared that compares the performance of three commercially available UPLC systems as they relate to the analysis of pharmaceutical compounds (73). A number of papers have been published that address general aspects of optical, electrochemical, and related detection in separation science. In one of these, the use of potassium permanganate and tris(2,2′-bipyridyl) ruthenium(III) chemiluminesence reagents for quantifying opium alkaloids has been reviewed (74). Information is provided in terms of their application in HPLC, capillary electrophoresis (CE) and related miniaturized devices, and flow injection analysis. In another paper, performance aspects of evaporative light scattering and charged aerosol detection have been discussed including problems of nonlinearity (75). A third paper reports on recent uses of electrochemical detection for assaying pharmaceuticals (76), a fourth on the developing spectral correlations of HPLC-diode array data between chromatographic runs as a means of impurity profiling (77), and a fifth the use of corona-charged aerosol detection for the supercritical fluid chromatography (SFC) analysis of various pharmaceuticals (78). The latter manuscript discusses how response differences of 2-3 are observable when different mobile phase compositions are used and how these differences can be minimized to a factor of 1.2-1.7 using a simple piece of hardware. The general use of packed bed SFC has again begin to increase for selected types of pharmaceutical applications such as those associated with the characterization of natural products (79) and polar and chiral compounds (80, 81). Column and eluent selection are discussed in these citations, as well as current applications of SFC singularly or in combination with other chromatographic methods. In addition to these papers, two have appeared that discuss the use of cyanopropyl modified silica as a useful phase for carrying out supercritical fluid chromatography separations (82) and optimization of the eluent composition when using a 2-ethylpyridine column (83). In the latter paper, the separation of neutral, acidic, and basic compounds with diverse physicochemical properties are discussed. Vibrational-based spectrometric techniques continue to gain in popularity as analytical tools to investigate various pharmaceutical problems, especially as they relate to formulation, content, and manufacturing. Three important reasons for this trend is the increasing availability of small dedicated in-line instruments, the techniques are nondestructive, and many types of measurements can be made on intact samples, especially those in the solid state. 4682
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On a relative basis and on the basis of the rapidly increasing numbers of papers being published, vibrational-based procedures, if not the fastest, are among the fastest growing methods in terms of their usage. Many reviews and papers dealing with general aspects of infrared, near-infrared, far-infrared, and Raman spectroscopies were published during the past 2 years. Because of the large number of them, only a sampling are included and discussed as representative examples of the types of work being carried out (84-115). One of the published papers presents a comprehensive overview of the use of infrared, near-infrared, and Raman to image pharmaceuticals (84), and four others discuss various aspects of near-infrared (85, 86), tetrahertz (87), and Raman (88) spectroscopies. Besides these papers, others have considered a variety of different calibration approaches (89-93), their use to characterize solids (94, 95), and imaging of the formulated products (84, 96-102). Likewise, many other papers were published that discussed specific aspects of a particular problem such as tablet identification (103), measuring the physical properties of the active compound and/or changes occurring in it during the manufacturing process (88, 104-107), product content (108-112), and the influence of molecular interactions, internal structure and/or coating on drug release kinetics (113-115). Specific examples for these types of studies that are related to individual products are discussed later in this review. However, some major areas of application for these techniques are for evaluating the physicochemical properties of the bulk drug substance and formulations containing it and physical changes occurring in the drug substance during the formulation process, polymorphic purity of the beginning and final product, and content monitoring both off-line and online. In the latter instance, they are being used to measure both the over amount and distribution of the active ingredients in the final formulation. A review has been published that discusses both uses and advantages of near-infrared spectroscopy as it applies to pharmaceutical analysis (116). As noted above, two important advantages of the technique are that it is nondestructive and samples can be assayed without or with only limited sample preparation. Two limitations of the technique are lack of sensitivity and the complexity of the spectra. Nevertheless, it is ideally suited for many types of process problems. Measuring hydration and water content are two important applications of the technique. Likewise, its use as a general identification tool is another important application. For many products, counterfeiting is a significant problem. In addition to vibrational spectroscopy, other spectrometric approaches are being used to study a wide range of pharmaceutical systems. One of these is confocal laser scanning microscopy (CLSM) especially as it relates to characterizing various emerging formulation and drug delivery materials (117). The cited reference discusses various applications of CLSM including phase-separated polymers, colloidal systems, microspheres, films and coatings, pellets, and tablets. Likewise, the use of CLSM for fluorescence imaging is discussed. Another technique, where its uses have expanded in scope, is solid-state nuclear magnetic resonance (SSNMR) spectroscopy. It is an increasingly important tool for characterizing the physical characteristics of drug formulations (118-124). These references discuss a variety of topics including
general aspects of SSNMR for studying pharmaceuticals, benchtop instrumentation and its use to investigate drug delivery, and the measurement of physical properties such as hydration and polymorphism. Although only a few examples are included in the current review, there are many publications that report on SSNMR studies concerned with various aspects of formulation. One of the driving forces behind the increasing use of SSNMR is the development of better drug delivery systems, which is an important goal for producing safer and more effective products. A good example of this is the development of custom designed nanomaterial for delivering anticancer agents to specific site/tissue locations. Interest in learning how the drug is being incorporated into the matrix and how and at what rate it is being released are important questions. In addition to the above, other reviews have appeared that consider recent advances in two-dimensional correlation spectroscopy (125), the use of NMR methods to screen cocrystallization as an emerging new class of solids for drug delivery (126), and general aspects of quantitative analysis by NMR (127). Much work is being carried out and reported, which is related to the use of cocrystallized materials to facilitate drug delivery. SEPARATION-BASED METHODOLOGY As discussed above, separation-based methodology continues to be the most often used approach for assaying pharmaceutical compounds with much of the standard assay work being carried out by reversed-phase HPLC and, to a much lesser extent, by some form of capillary electromigration (CEM) technique. In addition to the citations discussed previously in the General section, a variety of more specific reviews have been published that deal with topically related aspects of separation methodology. Among these are ones that discuss multicomponet analysis by HPLC (128), impurity determination using capillary electromigration methods (129), application of nonaqueous capillary electrophoresis (CE) to assay pharmaceuticals (130), recent developments of benzofurazan-based derivatizing reagents (131), and overviews of chiral analysis (132-134). In addition to more common separation methodology, in one of the latter reviews (132), the uses of other approaches are considered as costeffective alternatives to providing chiral information. Among those discussed are enantioselective membranes, amperometric biosensors, and molecularly imprinted polymers. Likewise, another one of the latter reviews (133) also considers the use of alternate chiral recognition tools like differing types of circular dichoric measurements. These, combined with traditionally used separation methods, provide a powerful “toolbox” of available approaches. General interest in CEM techniques seems to have stabilized based on a comparison of the number and scope of publications appearing over the last 2 years vs those published in past years. Nevertheless, they continue to be important techniques for measuring enantomeric purity (135, 136) and for characterizing more complex pharmaceuticals (i.e., natural products and proteinbased candidates) including their use in a microchip format using cyclodextrins (137) and ionic liquids (138). In the first of these papers, a four-channel electrophoresis chip platform is described that is capable of screening chiral selectors for their usefulness in carrying out enantiomeric assays. The total run time is less than 2 min for simultaneously screening four selectors. The cyclodextrins continue to be highly used chiral additives/selectors
for carrying out CE-based enantiomeric assays (139-141) as well as immobilized forms of them as stationary phases for performing similar HPLC separations. The latter paper discusses the use of a highly multiplexed CE system for carrying out many simultaneous chiral assays at once using R-, β-, and γ-cyclodextrins. Many other papers have been published that deal with various aspects of chiral separations (142-150). One of them is concerned with the enantioseparation techniques for andrenergic drugs (142), two deal with chiral separations by capillary electrophoresis (CE) (143, 144), and the others are concerned with sub- and supercritical fluid chromatography (145), simulated moving column SFC (146), microemulsion electrokinetic (MEK) separations (147, 148), and the use of chiral crown ethers (149) and ionic liquids in CE (150). The last two methods employ, respectively, (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and S-[3(chloro-2-hydroxypropyl)trimethylammonium) [bis(trifluoromethyl)sulfonyl)amide), which are reported to be easy to synthesize in a single step with readily available reagents. The latter reagent has been used for assaying a variety of different classes of pharmaceuticals including atenolol, propranolol, flurbiprofen, indoprofen, ibuprofen, ketoprofen, and warfarin. However, in some instances additional coselectors were needed depending on the structure and charge state of the analytes. In another review, the usefulness of vancomycin-related compounds as chiral selectors for their application in both HPLC and CE is discussed (151). Both practical and mechanistic aspects of the topic are considered. Similarly, numerous other papers have appeared that discuss particular chiral stationary phases, including cellulose tris(3,5-dichlorophenylcarbamate) modified silica (152), β-cyclodextrin chiral phases based on “click” chemistry (153), and R1 acid glycoprotein and ovomucoid protein phases (154). A stated advantage of the first of these phases is that it can be used with a wide range of commonly employed solvents and is rugged under various HPLC operating conditions. A second noted advantage is that it exhibits separation properties that are complementary to other materials. Representative examples of other chiral assays that have been designed for other specific compounds or groups of compounds are given in Table 1 (155-187). Although interest in developing new phases and additives continues to be high throughout the separation science community, much of the standard pharmaceutical methodology (i.e., excluding chiral separations) continues to employ relatively standard conditions both in terms of the phases and eluents being employed with the exception of the average particle size of the packings, which continues to decrease. Clearly there is growing interest in producing shorter columns packed with smaller particles
