Current and potential applications of simultaneous DSC-FTIR microspectroscopy for pharmaceutical analysis.

Quality control (QC) is the most important key issue in the pharmaceutical industry to ensure the quality of drug products. Many analytical instruments and techniques in pharmaceutical analysis are applied to assess the quality and quantity of the drugs. In the current and future trends, a combination of digitization, automation and hyphenation with high throughput on-line performance will be the topics for the future of pharmaceutical QC. The hyphenated analytical techniques have recently received great attention as unique means to solve complex analytical problems in a short period of time. This review article is an update on the recent potential applications of hyphenated technique developed from the coupling of a rapid separation or induction technique (differential scanning calorimetry; DSC) and an on-line spectroscopic (Fourier transform infrared; FTIR) detection technology to carry out an one-step solid-state analysis in pharmaceutical formulation developments, including (1) intramolecular condensation of pharmaceutical polymers, (2) intramolecular cyclization of drugs and sweetener, (3) polymorphic transformation of drugs and excipients, (4) drug-polymer (excipient) interaction, (5) fast cocrystal screening and formation. This simultaneous DSC-FTIR microspectroscopy can also provide an easy and direct method for one-step screening and qualitative detection of drug stability in real time.

Thermoresponsive gating membranes embedded with liquid crystal(s) for pulsatile transdermal drug delivery: An overview and perspectives.

Due to the circadian rhythm regulation of almost every biological process in the human body, physiological and biochemical conditions vary considerably over the course of a 24-h period. Thus, optimal drug delivery and therapy should be effectively controlled to achieve the desired therapeutic plasma concentrations and therapeutic drug responses at the required time according to chronopharmacological concepts, rather than continuous maintenance of constant drug concentrations for an extended time period. For many drugs, it is not always necessary to constantly deliver a drug into the human body under disease conditions due to rhythmic variations. Pulsatile drug delivery systems (PDDSs) have been receiving more attention in pharmaceutical development by providing a predetermined lag period, followed by a fast or rate-controlled drug release after application. PDDSs are characterized by a programmed drug release, which may release a drug at repeatable pulses to match the biological and clinical needs of a given disease therapy. This review article focuses on thermoresponsive gating membranes embedded with liquid crystals (LCs) for transdermal drug delivery using PDDS technology. In addition, the principal rationale and the advanced approaches for the use of PDDSs, the marketed products of chronotherapeutic DDSs with pulsatile function designed by various PDDS technologies, pulsatile drug delivery designed with thermoresponsive polymers, challenges and opportunities of transdermal drug delivery, and novel approaches of LC systems for drug delivery are reviewed and discussed. A brief overview of all academic research articles concerning single LC- or binary LC-embedded thermoresponsive membranes with a switchable on-off permeation function through topical application by an external temperature control, which may modulate the dosing interval and administration time according to the therapeutic needs of the human body, is also compiled and presented. In the near future, since thermal-based approaches have become a well-accepted method to enhance transdermal delivery of different water-soluble drugs and macromolecules, a combination of the thermal-assisted approach with thermoresponsive LCs membranes will have the potential to improve PDDS applications but still poses a great challenge.

Assessment of Unicuspid Aortic Valve Stenosis Using Multimodality Imaging, X-ray Radiography and Raman Analysis.

Unicuspid aortic valve (UAV) is an extremely rare form of congenital cardiac malformation, leading to aortic stenosis (AS), aortic regurgitation (AR), or both. We report the case of a 55-year-old man with unicommissural UAV associated with severe AS and mild AR using different multimodality imaging approaches. The excised UAV isolated after aortic valve replacement exhibited an eccentric "teardrop" opening with a slit-shaped unicommissural structure. Raman spectroscopic results indicated that 3 unevenly distributed components were deposited on the surface of the UAV, in which calcium hydroxyapatite and type-B carbonate apatite were the predominate components deposited on the surface, leading to severe AS formation.

Echocardiographic manifestations and chemical composition of stenotic bicuspid aortic valves.

Bicuspid aortic valve (BAV) is an inherited form of heart disease with only two aortic valve leaflets via a disorder of cardiac valvulogenesis. We investigated the in vivo echocardiographic features of cardiac morphology in patients with BAV and the ex vivo compositional components of all the excised BAV leaflets isolated from BAV patients. Three BAV patients were randomly selected. All patients underwent 2D transthoracic echocardiography (TTE) with a Doppler ultrasound tool. The compositional components of each respective BAV leaflet for all the excised BAVs were determined by a portable fiber-optic Raman spectroscopy. Preoperative TTE revealed the thickened and calcified BAV leaflets, and stenotic aortic flow for all BAV patients. These BAV patients exhibited severe aortic stenosis (AS) by the lower values of aortic valve area (AVA) index. One patient showed a more significant left ventricle hypertrophy, whereas two patients exhibited a significant aortic regurgitation (AR). In addition, three different Raman spectral patterns were summed up from 121 randomized Raman determinations for all the excised BAV leaflets. The main calcified deposition in each BAV leaflet was formed by large amounts of calcium hydroxyapatite and type-B carbonate apatite (Raman bands at 960 and 1070 cm-1). The calcified BAV leaflets were composed of different compositional components such as calcium hydroxyapatite, type-B carbonate apatite, lipids, proteins, cholesterol and ß-carotene. The rare NL subtype of type 1 BAV morphotype was found in one patient, but two patients had the purely BAV morphotype with two equal-sized leaflets.

New morphological classification of congenital quadricuspid aortic valve and its histopathologic features.

We report a 52-year-old male patient who had a quadricuspid aortic valve (QAV) associated with aortic regurgitation (AR) and left ventricular hypertrophy (LVH). A new accessory cusp (ACC) with maximum thickness than other cusps was located between right coronary cusp (RCC) and left coronary cusp (LCC). The histopathological features revealed markedly thickened and distorted cusp architecture with fibrosis and/or myxomatous degeneration in both non-coronary cusp (NCC) and ACC. Two equal sizes for larger cusps (RCC and NCC) and two equal sizes for smaller cusps (LCC and ACC) were obtained. This QAV belonged to type C QAV of Hurwitz's classification, but also suggested as a modified type III of Jagannath's classification or a new type V of Nakamura's classification by locating ACC between RCC and LCC.

Non-isothermal dehydration kinetic study of aspartame hemihydrate using DSC, TGA and DSC-FTIR microspectroscopy.

Three thermal analytical techniques such as differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) using five heating rates, and DSC-Fourier Transform Infrared (DSC-FTIR) microspectroscopy using one heating rate, were used to determine the thermal characteristics and the dehydration process of aspartame (APM) hemihydrate in the solid state. The intramolecular cyclization process of APM anhydrate was also examined. One exothermic and four endothermic peaks were observed in the DSC thermogram of APM hemihydrate, in which the exothermic peak was due to the crystallization of some amorphous APM caused by dehydration process from hemihydrate to anhydride. While four endothermic peaks were corresponded to the evaporation of absorbed water, the dehydration of hemihydrate, the diketopiperazines (DKP) formation via intramolecular cyclization, and the melting of DKP, respectively. The weight loss measured in TGA curve of APM hemihydrate was associated with these endothermic peaks in the DSC thermogram. According to the Flynn-Wall-Ozawa (FWO) model, the activation energy of dehydration process within 100-150 °C was about 218 ± 11 kJ/mol determined by TGA technique. Both the dehydration and DKP formation processes for solid-state APM hemihydrate were markedly evidenced from the thermal-responsive changes in several specific FTIR bands by a single-step DSC-FTIR microspectroscopy.

Ex vivo assessment of valve thickness/calcification of patients with calcific aortic stenosis in relation to in vivo clinical outcomes.

Calcific aortic stenosis (AS) plays a critical role in the risk of cardiovascular disease. This preliminary study examined the relationship between the ex vivo valve thickness/calcification and in vivo clinical outcomes of Chinese patients with calcific AS. Six Chinese patients (3 patients with tricuspid aortic valves (TAV)) and 3 patients with. bicuspid aortic valves (BAV) with calcific AS undergoing heart valve replacement were initially chosen for this study. In vivo medical imaging of these calcific AS patients was evaluated using computed tomography and echocardiography. The ex vivo measurements including the actual thickness, calcified area and components of the calcified aortic values excised were performed by a digimatic caliper, X-ray equipment with a cellSens imaging analysis and portable Raman spectroscopy, respectively. Six patients were diagnosed with symptomatic moderate-to-severe AS. The thickness of noncoronary (N) leaflet in the excised TAV was significantly thicker than left-coronary (L) leaflet (p < 0.01), and right-coronary (R) leaflet was also thicker than L (p < 0.05), but no significant difference occurred between N and R (p > 0.05). The extent of calcification in the excised TAV was a statistically significant difference between N and L (p < 0.01) and between R and L (p < 0.01), respectively. However, there was no significant difference between R and L in both thickness and calcification for the excised BAV (p > 0.05). The patients No. 1-3 were found to be TAV with partial commissural fusion. The patient No. 4 was classified as a type 1 NL-BAV morphotype, but both patients 5 and 6 were found to be true BAV (type 0 lateral-BAV). Each calcified valve leaflet was composed of apatites, proteins (collagen and proteoglycan), and a small amount of ß-carotene and cholesterol after Raman spectral determination. The calcified nodules of each valve leaflet were predominately identified to be calcium hydroxyapatite and type-B carbonate apatite. However, octacalcium phosphate was also detected in the protein-rich part of calcified valve leaflets. A positive correlation was observed between thickness and calcification for both excised TAV and BAV after ex vivo examinations. Moreover, a negative relationship was obtained among in vivo AVA index, ex vivo thickness and ex vivo calcification for these calcific AS patients.

Simultaneous screening and detection of pharmaceutical co-crystals by the one-step DSC-FTIR microspectroscopic technique.

Various methods and analytical techniques for the preparation and identification of pharmaceutical co-crystals have been applied, but these operations require considerable time for the screening and preparation of co-crystals. In this review, a powerful method that combines Fourier-transform infrared (FTIR) microspectroscopy with thermal analysis is introduced. This unique one-step real-time differential scanning calorimetry (DSC)-FTIR microspectroscopic approach has been successfully applied to simultaneously and directly screen and detect pharmaceutical co-crystal formation in systems such as indomethacin-saccharin, indomethacin-nicotinamide, carbamazepine-glutaric-acid, metaxalone-succinic-acid and piroxicam-saccharin. This powerful one-step DSC-FTIR combined technique provides an easy and direct method for one-step screening and qualitative detection of co-crystal formation in real time.

Mechanochemical Approaches to Pharmaceutical Cocrystal Formation and Stability Analysis.

Solid-state mechanochemical grinding is important for promoting cocrystal formation, particularly in the design of new solids in the pharmaceutical industry. Pharmaceutical cocrystals are defined as crystalline materials comprising an active pharmaceutical ingredient (API) and one or more appropriate coformers in a definite stoichiometric ratio, formed via non-covalent interactions. Recently, both the US FDA (2013) and the EU EMA (2015) provided a Guidance for Industry and a Reflection Paper, respectively, emphasizing that cocrystals are a new type of substance with potential applications in the pharmaceutical industry. This paper contains a brief and systematic overview of pharmaceutical cocrystals prepared by four grinding processes: neat grinding, solvent-assisted grinding, thermal stress after neat grinding, and polymer-assisted grinding. The paper also highlights some examples of pharmaceutical cocrystals prepared by the above grinding approaches, and discusses the stability of cocrystals prepared by mechanical grinding. Also, an overview of cocrystals that are commercially available or undergoing clinical trials is given. A novel methodology for real-time and in situ monitoring of mechanochemical grinding reactions using various analytical techniques is addressed and can be expected to be applied in the near future.

Spectral and morphological classification of different chronic and acute Taiwanese gallstones via FTIR, SEM and ESEM-EDX microanalyses.

BACKGROUND: Gallstone disease is one of the leading upper gastrointestinal surgical problems in different countries. AIMS: To analyze the chronic gallstones and acute gallbladder sludge retrieved from 36 Taiwanese patients. METHODS: FTIR microspectroscopy was used to classify the types of gallstones, and an ESEM-EDX microanalysis was first applied to determine the microstructural features and elemental compositions of the various gallstones. Bacteria presented on the surface of gallstones were also detected by SEM. RESULTS: Four types of gallstones were obtained from these 36 Taiwanese patients: calcium bilirubinate (CaBR) stones (30.6%), cholesterol stones (19.4%), mixed stones including 6 subtypes (47.2%), and acute gallbladder sludge (2.8%) made of CaBR and protein/insoluble biomaterials. Bacteria imprints and bacterial discharges or bacterial biofilms were also found on the surface of gallstones and acute sludge under a SEM observation. ESEM-EDX results revealed that calcium was found to be the main constituent of all of the types of stones except cholesterol stones, and aluminum was also presented in most of the stones and sludge samples. Chloride was only detected in the acute gallbladder sludge. CONCLUSION: FTIR spectra, morphological features, and elemental compositions of the acute gallbladder sludge were different from those of the chronic gallstones.

Dual-Energy Computed Tomography Gemstone Spectral Imaging: A Novel Technique to Determine Human Cardiac Calculus Composition.

OBJECTIVE: Understanding the chemical composition of any calculus in different human organs is essential for choosing the best treatment strategy for patients. The purpose of this study was to assess the capability of determining the chemical composition of a human cardiac calculus using gemstone spectral imaging (GSI) mode on a single-source dual-energy computed tomography (DECT) in vitro. METHODS: The cardiac calculus was directly scanned on the Discovery CT750 HD FREEdom Edition using GSI mode, in vitro. A portable fiber-optic Raman spectroscopy was also applied to verify the quantitative accuracy of the DECT measurements. RESULTS: The results of spectral DECT measurements indicate that effective Z values in 3 designated positions located in this calculus were 15.02 to 15.47, which are close to values of 15.74 to 15.86, corresponding to the effective Z values of calcium apatite and hydroxyapatite. The Raman spectral data were also reflected by the predominant Raman peak at 960 cm for hydroxyapatite and the minor peak at 875 cm for calcium apatite. CONCLUSIONS: A potential single-source DECT with GSI mode was first used to examine the morphological characteristics and chemical compositions of a giant human cardiac calculus, in vitro. The CT results were consistent with the Raman spectral data, suggesting that spectral CT imaging techniques could be accurately used to diagnose and characterize the compositional materials in the cardiac calculus.

One-step Real-time Food Quality Analysis by Simultaneous DSC-FTIR Microspectroscopy.

This review discusses an analytical technique that combines differential scanning calorimetry and Fourier-transform infrared (DSC-FTIR) microspectroscopy, which simulates the accelerated stability test and detects decomposition products simultaneously in real time. We show that the DSC-FTIR technique is a fast, simple and powerful analytical tool with applications in food sciences. This technique has been applied successfully to the simultaneous investigation of: encapsulated squid oil stability; the dehydration and intramolecular condensation of sweetener (aspartame); the dehydration, rehydration and solidification of trehalose; and online monitoring of the Maillard reaction for glucose (Glc)/asparagine (Asn) in the solid state. This technique delivers rapid and appropriate interpretations with food science applications.

Thermoanalytical and Fourier transform infrared spectral curve-fitting techniques used to investigate the amorphous indomethacin formation and its physical stability in Indomethacin-Soluplus® solid dispersions.

The amorphous form of a drug has higher water solubility and faster dissolution rate than its crystalline form. However, the amorphous form is less thermodynamically stable and may recrystallize during manufacturing and storage. Maintaining the amorphous state of drug in a solid dosage form is extremely important to ensure product quality. The purpose of this study was to quantitatively determine the amount of amorphous indomethacin (INDO) formed in the Soluplus® solid dispersions using thermoanalytical and Fourier transform infrared (FTIR) spectral curve-fitting techniques. The INDO/Soluplus® solid dispersions with various weight ratios of both components were prepared by air-drying and heat-drying processes. A predominate IR peak at 1683cm(-1) for amorphous INDO was selected as a marker for monitoring the solid state of INDO in the INDO/Soluplus® solid dispersions. The physical stability of amorphous INDO in the INDO/Soluplus® solid dispersions prepared by both drying processes was also studied under accelerated conditions. A typical endothermic peak at 161°C for γ-form of INDO (γ-INDO) disappeared from all the differential scanning calorimetry (DSC) curves of INDO/Soluplus® solid dispersions, suggesting the amorphization of INDO caused by Soluplus® after drying. In addition, two unique IR peaks at 1682 (1681) and 1593 (1591)cm(-1) corresponded to the amorphous form of INDO were observed in the FTIR spectra of all the INDO/Soluplus® solid dispersions. The quantitative amounts of amorphous INDO formed in all the INDO/Soluplus® solid dispersions were increased with the increase of γ-INDO loaded into the INDO/Soluplus® solid dispersions by applying curve-fitting technique. However, the intermolecular hydrogen bonding interaction between Soluplus® and INDO were only observed in the samples prepared by heat-drying process, due to a marked spectral shift from 1636 to 1628cm(-1) in the INDO/Soluplus® solid dispersions. The INDO/Soluplus® solid dispersions prepared by both drying processes could keep the amorphous state of INDO in the INDO/Soluplus® solid dispersions at the accelerated storage condition.

Molecular perspectives on solid-state phase transformation and chemical reactivity of drugs: metoclopramide as an example.

Here, I provide an overview of the solid-state characteristics, phase transformations and chemical reactions of metoclopramide hydrochloride monohydrate (MCP HCl H2O). Three unique techniques, including thermoanalytical methods, one-step simultaneous differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) microspectroscopy, and hot-stage microscopic (HSM) imaging, have been applied to study the solid-state phase transitions of MCP HCl H2O in continuous dehydration, amorphization and recrystallization processes. I also review the effects of grinding or heating on ion-exchange reactions, milling, compression or colyophilization on Maillard reactions, and γ-ray irradiation or electron beams on radiolysis in the solid state. I also report the exposure of MCP HCl H2O in solution to light, irradiation, oxidants or π-acceptors. This review will serve as a useful keynote for the evolving realm of solid-state chemistry research.

An investigation of indomethacin-nicotinamide cocrystal formation induced by thermal stress in the solid or liquid state.

The impact of thermal stress on indomethacin (IMC)-nicotinamide (NIC) cocrystal formation with or without neat cogrinding was investigated using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) microspectroscopy, and simultaneous DSC-FTIR microspectroscopy in the solid or liquid state. Different evaporation methods for preparing IMC-NIC cocrystals were also compared. The results indicated that even after cogrinding for 40 min, the FTIR spectra for all IMC-NIC ground mixtures were superimposable on the FTIR spectra of IMC and NIC components, suggesting there was no cocrystal formation between IMC and NIC after cogrinding. However, these IMC-NIC ground mixtures appear to easily undergo cocrystal formation after the application of DSC determination. Under thermal stress induced by DSC, the amount of cocrystal formation increased with increasing cogrinding time. Moreover, simultaneous DSC-FTIR microspectroscopy was a useful one-step technique to induce and clarify the thermal-induced stepwise mechanism of IMC-NIC cocrystal formation from the ground mixture in real time. Different solvent evaporation rates induced by thermal stress significantly influenced IMC-NIC cocrystal formation in the liquid state. In particular, microwave heating may promote IMC-NIC cocrystal formation in a short time.

Biochemical and molecular aspects of spectral diagnosis in calcinosis cutis.

Calcinosis cutis (CC) is a type of calcinosis wherein insoluble compounds or salts deposited on the skin. Clinical diagnosis of CC is usually achieved through time consuming histopathological or immunohistochemical procedures, but it can only be empirically identified by experienced practitioners. The use of advanced vibrational spectroscopy has been recently shown to have great potential as a diagnostic technique for various diseased tissues because it analyses the chemical composition of diseased tissue rather than its anatomy and predicts disease progression. This review article includes a summary of the application of Fourier transform infrared (FT-IR) and Raman spectroscopic or microspectroscopic analysis for the rapid diagnosis and identification of the chemical composition of skin calcified deposits in patients with various CC symptoms. Both advanced techniques not only can detect the types of insoluble salts such as calcium phosphate, calcium carbonate, and monosodium urate, and ß-carotene in the calcified deposits of human skin tissue but also can directly differentiate the carbonate substitution in the apatite structure of the skin calcified deposits. In particular, the combination of both vibrational techniques may provide complementary information to simultaneously assess the intact components of the calcified deposits. In the future, both FT-IR and Raman vibrational microspectroscopic techniques will become available tools to support the standard test techniques currently used in some clinical diagnoses. Molecular spectroscopy technique is rapidly changing disease diagnosis and management.

An overview of famotidine polymorphs: solid-state characteristics, thermodynamics, polymorphic transformation and quality control.

Crystal polymorphism of pharmaceuticals has well-known profound effects on the physical, chemical, and pharmaceutical properties of drugs, which can result in changes in the solubility, stability, dissolution, bioavailability, and efficacy of drugs. In this review article, famotidine (FAM), which has a well-known trade name of Pepcid®, was selected as a model drug. Although FAM has three polymorphs (forms A, B and C), forms A and B have been commonly discussed. The active pharmaceutical ingredient (API) in the commercial version of FAM is the metastable form B. FAM has been a concern of FDA because of the physical properties, solubilities, bioavailabilities, or bioequivalencies of the different polymorphic forms. In addition, a patent infringement suit of FAM polymorph had been made sound legal arguments in the pharmaceutical market. We review the solid-state characteristics, thermodynamics, polymorphic transformation, and quality control of FAM in drug products. In particular, pharmaceutical processes, such as grinding, compression, and heating temperature have a significant effect on the polymorphic transformation of FAM. Moreover, environmental humidity and residual water content should be well controlled to prevent polymorphic transformation of FAM during pharmaceutical processing. Several thermal and spectroscopic analytical techniques used for qualitative and quantitative determinations of polymorphic transformation of FAM after different treatments or quality control of FAM in the commercial tablets before and after the expiration dates have been discussed.

Application of scanning electron microscopy and X-ray microanalysis: FE-SEM, ESEM-EDS, and EDS mapping for studying the characteristics of topographical microstructure and elemental mapping of human cardiac calcified deposition.

To explore the pathogenic mineral formation in a huge cardiolith isolated from the left heart atrium of an 80-year-old male patient, field emission scanning electron microscopy (FE-SEM) was used to analyze the topographic microstructure and perform elemental mapping in a cross-section of the cardiac calcified deposit after dissection. Environmental SEM equipped with an energy dispersive X-ray spectrometer (EDS) was also used to investigate the composition and spatial distribution of elements in the cross-section, and fiberoptic Raman spectroscopy was used to reidentify the chemical composition of designated positions. The results indicated that calcium hydroxyapatite and cholesterol were the main components of the cardiac calculus. The plate-like structures of calcium hydroxyapatite were unevenly spread over the cholesterol of the cardiac calculus. The calcium hydroxyapatite-rich area exhibited higher amounts of C, O, P, and Ca elements as well as trace amounts of N, Na, Mg, and Al, whereas the major concentration of C, minor concentrations of N and O, and trace amounts of P and Ca were observed in the cholesterol-rich area. Hypercholesterolemia associated with calcification of this cardiac calculus was proposed. Both FE-SEM and ESEM energy dispersive X-ray microanalyses were performed directly, for the first time, to provide useful information on the microstructural characteristics and spatial distribution of elements on the surface of human cardiac calculi.

Composition and distribution of elements and ultrastructural topography of a human cardiac calculus.

Trace elements (TEs) may contribute to the formation of calculi or stones or be involved in the aetiopathogenesis of stone diseases. The compositions and spatial distribution of elements from the inner nucleus to outer crust of the cardiac calculus were investigated by energy-dispersive X-ray fluorescence (EDXRF) spectrometer. The surface topograph, distribution map of elements, elemental and chemical compositions were also determined by environmental scanning electron microscope (ESEM)-energy-dispersive X-ray (EDX) analysis. Twenty-five elements were identifiable from 18 positions on the cardiac calculus by EDXRF spectrometer, in which the highest concentrations of toxic TEs (Ni, Pt, Hg, Sn, Pb, W, Au, Al, Si) and higher levels of essential TEs (Ca, Sr, Cr, P) were detected. A moderate positive Pearson's correlation between TEs concentrations of Mg, Ca or P and location differences from centre to periphery in the cardiac calculus was observed. A positive correlation was also found for Ca/Zn and Ca/Cu, indicating the gradual increase of calcium concentration from inner nucleus to outer crust of cardiac calculus. The drop-like nodules/crystals on the surface of petrous part of cardiac calculus were observed from ESEM analysis. ESEM-EDX analysis determined the calculus to be predominantly composed of calcium hydroxyapatite and cholesterol, as indicated by the petrous surface and drop-like nodules/crystals, respectively. This composition was confirmed using a portable Raman analyser. The spatial distribution analysis indicated a gradual increase in Mg, P and Ca concentrations from the inner nucleus to the outer crust of the cardiac calculus. The major chemical compositions of calcium hydroxyapatite and cholesterol were detected on this cardiac calculus.