Manuel Holcomb

Sample preparation for the analysis of flavors and off-flavors in foods

Off-flavors in foods may originate from environmental pollutants, the growth of microorganisms, oxidation of lipids, or endogenous enzymatic decomposition in the foods. The chromatographic analysis of flavors and off-flavors in foods usually requires that the samples first be processed to remove as many interfering compounds as possible. For analysis of foods by gas chromatography (GC), sample preparation may include mincing, homogenation, centrifugation, distillation, simple solvent extraction, supercritical fluid extraction, pressurized-fluid extraction, microwave-assisted extraction, Soxhlet extraction, or methylation. For high-performance liquid chromatography of amines in fish, cheese, sausage and olive oil or aldehydes in fruit juice, sample preparation may include solvent extraction and derivatization. Headspace GC analysis of orange juice, fish, dehydrated potatoes, and milk requires almost no sample preparation. Purge-and-trap GC analysis of dairy products, seafoods, and garlic may require heating, microwave-mediated distillation, purging the sample with inert gases and trapping the analytes with Tenax or C18, thermal desorption, cryofocusing, or elution with ethyl acetate. Solid-phase microextraction GC analysis of spices, milk and fish can involve microwave-mediated distillation, and usually requires adsorption on poly(dimethyl)siloxane or electrodeposition on fibers followed by thermal desorption. For short-path thermal desorption GC analysis of spices, herbs, coffee, peanuts, candy, mushrooms, beverages, olive oil, honey, and milk, samples are placed in a glass-lined stainless steel thermal desorption tube, which is purged with helium and then heated gradually to desorb the volatiles for analysis. Few of the methods that are available for analysis of food flavors and off-flavors can be described simultaneously as cheap, easy and good.

Determination of aflatoxins in food products by chromatography

Several chromatographic methods for the determination of aflatoxins in agricultural and food products are reviewed. During the past two decades, identification and determination of aflatoxins were done by thin-layer chromatography (TLC) because it was easy, fast and inexpensive. However, high-performance liquid chromatography (HPLC) using fluorescence detection is now the method of choice for determining aflatoxins and is also growing in popularity for their identification. The reasons for selecting HPLC over TLC can be summarized as the ability to analyze for a wide variety of compounds, including compounds that are easily degraded by heat, light or air, the ease of adaptation to confirmatory procedures, the potential for automation and the dramatic improvement in instrumentation, including the development of increasingly sensitive fluorescence and electrochemical detectors and short, high-resolution, reversed-phase columns.

SFE extraction of aflatoxins (B1, B2, G1, and G2) from corn and analysis by HPLC

A supercritical-fluid extraction (SFE) method has been developed that extracts aflatoxins (B1, B2, G1 and G2) from spiked corn using modified supercritical carbon dioxide. Methanol is added to the SFE extraction cell containing the corn layered between Hydromatrix (a dispersing material) which helps to prevent the clogging of the frits and reduces the effect of moisture on the extraction. The corn is held in static extraction at 65°C and 51.7 MPa for 15 min followed by a dynamic extraction with 20 mL of liquid carbon dioxide. The sample is depressurized and modifier re-added with a 10-min static extraction at 51.7 MPa followed by a dynamic extraction with 20 mL of liquid carbon dioxide. The SFE extract is collected in 10 mL of chloroform and further cleaned up with a Florisil Sep-Pak. The aflatoxins were analyzed by HPLC using fluorescence detection after post-column derivatization with iodine. Recoveries of the aflatoxins B1, B2, G1, and G2) over a range of 3 to 11 ng g-1 averaged 77.3, 82.9, 75.4, and 80.3%, respectively.

Defining and using microbial spectral databases

This work shows how fingerprints of mass spectral patterns from microbial isolates are affected by variations in instrumental condition, by sample environment, and by sample handling factors. It describes a novel method by which pattern distortions can be mathematically corrected for variations in factors not amenable to experimental control. One uncontrollable variable is “between-batch” differences in culture media. Another, relevant for determination of noncultured extracts, is differences between the cells’ environmental experience (e.g., starved environmental extracts versus cultured standards). The method suggests that, after a single growth cycle on a solid medium (perhaps, a selective one), pyrolysis MS spectra of microbial isolates can be algorithmically compensated and an unknown isolate identified using a spectral database defined by culture on a different (perhaps, nonselective) medium. This reduces identification time to as few as 24 h from sample collection. The concept also proposes a possible way to compensate certain noncultured, nonisolated samples (e.g., cells concentrated from urine or impacted from aerosol or semi-selectively extracted by immuno-affinity methods from heavily contaminated matrices) for identification within half an hour. Using the method, microbial mass spectra from different labs can be assembled into coherent databases similar to those routinely used to identify pure compounds. This type of data treatment is applicable for rapid detection in biowarfare and bioterror events as well as in forensic, research, and clinical laboratory contexts.

HPLC with Electrochemical and Fluorescence Detection of the OPA/2-Methyl-2-propanethiol Derivative of Fumonisin B1

Abstract The o-phthalaldehyde (OPA) derivative of fumonisin B1 was prepared in the presence of 2-methyl-2-propanethiol (tert-butyl thiol). A hydrodynamic voltammogram for the derivative indicated that the optimum voltage for maximum electrochemical response was ±0.7 V. The electrochemical response of the OPA/tert-butyl derivative was unstable. However, the fluorescence response was found to be stable for over an hour after an initial 30 minute reaction time. The minimum detectable limit (MDL) of the OPA/tert-butyl derivative using fluorescence detection was 30 ng/ml as compared to 250 ng/ml for electrochemical detection.