Mark R. Witkowski

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

A multidisciplinary approach for the analysis of an adulterated dietary supplement where the active pharmaceutical ingredient was embedded in the capsule shell.

Fourier transform infrared (FT-IR) microspectroscopic imaging, Raman microspectroscopy, optical microscopy and high performance liquid chromatography with mass spectrometric (LC/MS) detection were employed to examine a dietary supplement adulterated with an undeclared active pharmaceutical ingredient (API). While a trace level of the API was detected in the capsule contents, a higher concentration of API was found in the capsule shell, which indicated the use of an unconventional manufacturing process to hide the API and thus avoid detection. This study demonstrates the need for a multidisciplinary approach to provide a complete assessment of a suspect adulterated dietary supplement.

Analysis of Counterfeit Pharmaceutical Tablet Cores Utilizing Macroscopic Infrared Spectroscopy and Infrared Spectroscopic Imaging

Advantages and limitations of analyzing authentic and counterfeit pharmaceutical tablets with both macro (nonimaging) attenuated total internal reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and micro ATR-FT-IR spectroscopic imaging have been evaluated. The results of this study demonstrated that micro ATR imaging was more effective for extracting formulation information (sourcing), whereas a macro ATR approach was better suited for counterfeit detection (screening). More importantly, this study demonstrated that a thorough analysis of the counterfeit core can be achieved by combining the results of both techniques.

GHB free acid: I. Solution formation studies and spectroscopic characterization by 1HNMR and FT-IR.

ABSTRACT: In forensic evidence, γ‐hydroxybutyric acid (GHB) has frequently been encountered in one of its salt forms (γ‐hydroxybutyrate), but has also been encountered in its free acid form (GHB). Owing to the physical properties, encounters of the free acid have been largely restricted to forensic exhibits comprising aqueous solutions, such as acidic beverages that have been “spiked” or formulated with GHB salts or γ‐butyrolactone (GBL). The analysis of GHB free acid presents particular difficulties including the potential for altering the original proportions of GHB free acid, GHB carboxylate, and GBL in the course of analysis, and discrimination between GHB free acid and carboxylate forms. In this work, the formation of GHB free acid in aqueous solutions (water and/or D2O) was studied as a function of solution pH. Proton nuclear magnetic resonance (1HNMR) and Fourier‐transform infrared spectrometry (FT‐IR) measurements were obtained on freshly prepared mixtures of NaGHB and HCl stock solutions representing a series of points along the GHB titration curve. Both 1HNMR and FT‐IR were shown to track the changing proportions of GHB free acid and carboxylate forms as a function of pH, while simultaneously monitoring for the formation of the lactone (GBL). The results were consistent with acid–base conversion behavior for a carboxylic acid. 1HNMR was shown to provide an ideal means for analysis of aqueous‐based GHB/GBL forensic exhibits based on simple dilution of the neat liquid exhibit, without altering the original proportions of GHB free acid, carboxylate, and GBL in the samples.

Rapid Molecular Imaging Using Attenuated Total Internal Reflection Planar Array Infrared Spectroscopy for the Analysis of Counterfeit Pharmaceutical Tablets

A planar array infrared (PA-IR) spectrograph containing an attenuated total internal reflection (ATR) accessory has been constructed in order to permit rapid analysis of poorly transmitting materials. The technique has been optimized to allow molecular spectroscopic information to be collected in roughly 2 seconds with a corresponding peak-to-peak noise value as low as 2.14 × 10−4 absorbance units. Additionally, up to 150 spectra could be extracted from sample sizes as large as 6 mm where each spatial element measured 40 × 200 μm at the sample position. An application study for this technique entailed developing an embedding method that allows cross-sectioned pharmaceutical tablets to be brought into intimate contact with the internal reflection element (IRE) of the accessory. A supplemental investigation involved calculating the yield strength of multiple IRE materials in order to determine the maximum amount of pressure that can be applied to a sample without damaging the IRE. Finally, feasibility was demonstrated for using the instrument/accessory as a means to rapidly authenticate suspected counterfeit pharmaceutical tablets.

GHB Free Acid: II. Isolation and Spectroscopic Characterization for Forensic Analysis

ABSTRACT: A reference standard for γ‐hydroxybutyric acid (GHB) free acid is not commercially available, making its analysis in forensic exhibits more difficult. GHB free acid is typically encountered in aqueous solution and in the presence of the lactone, γ‐butyrolactone (GBL), presenting difficulty in Fourier transform infrared (FT‐IR) analysis. The strong infrared (IR) absorptivity of the GBL carbonyl band, the shifting of the GBL carbonyl band in aqueous solutions, and the position of the O–H bend for water can mask the main carbonyl band for GHB free acid. Model solutions of β‐hydroxybutyric acid (BHB) and GBL were studied in order to further understand the masking of the GHB free acid carbonyl band in FT‐IR analysis. The use of second derivative FT‐IR spectroscopy was shown to provide resolution of the free acid carbonyl band, and a presumptive test for GHB free acid was developed and applied. An extension of this work included preparing, for use as a standard reference material, small amounts (≤10 mg) of GHB free acid. Preparation was based on the instantaneous reaction of GHBs sodium salt with a stoichiometric amount of hydrochloric acid in aqueous solution, and subsequent isolation of the free acid in neat liquid form. Both FT‐IR and proton nuclear magnetic resonance spectra of the neat reference material were obtained and used to verify its identity. The isolation of GHB free acid from actual forensic exhibits is also presented, with identity confirmation using FT‐IR.

Analysis of Hypodermic Needles and Syringes for the Presence of Blood and Polydimethylsiloxane (Silicone) Utilizing Microchemical Tests and Infrared Spectroscopy

Suspect hypodermic needles and syringes were seized from an unlicensed individual who was allegedly injecting patients with silicone (polydimethylsiloxane [PDMS]) for cosmetic enhancement. Since control syringe barrels and needles often contain an interfering PDMS lubricant, a risk for false positives of foreign PDMS exists. The focus of this report was to minimize this risk and determine a quick and reliable test for the presence of blood in PDMS matrices. Using ATR‐FT‐IR spectroscopy, the risk for false‐positive identification of foreign PDMS was reduced by (i) overfilling the sampling aperture to prevent spectral distortions and (ii) sampling a region of the suspect syringe/needle assembly where manufacturer‐applied PDMS is not typically located. Analysis for blood indicated that the Teichman microchemical test was effective for detecting blood in the presence of PDMS. Overall, detecting PDMS established intent and detecting blood established that the needle containing the PDMS had been used for injection.

Characterization of Resultant Micro Chemical Test Crystalline Formations using Optical, Fourier Transform Infrared (FT-IR) and Raman Microscopies

Micro chemical tests have been used to aid in the identification of materials and as rapid screening tools for microscopists and forensic chemists for decades. It is generally understood in the microscopy community that these types of tests, with unique resultant crystalline formations, can be quite specific and used for material identification. However, some consider micro chemical tests as secondary identification tests, since many of the tests involve the same chemical reagent used for a variety of analytes. Many of these micro chemical tests are used at the FCC in the analysis of forensic evidence to rapidly determine the presence or absence of drugs and other organic and inorganic chemicals. These tests can be very useful in the characterization of dietary supplements, which consist of very complex mixtures. It can be argued that the need for micro chemical tests is becoming less important with the availability of very sensitive instrumental techniques such as mass spectrometry. However, these tests allow for very rapid separation of a heterogeneous mixture, provide sample matrix information and information on the crystallinity of a material. This is especially important for pharmaceuticals, as a difference in drug crystallinity can affect its bioavailability.