Bacterial analysis by MALDI-TOF mass spectrometry: an inter-laboratory comparison.

Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed.

Assessing the toxicity of polymeric food-contact substances.

The US Food and Drug Administration's Office of Food Additive Safety in the Center for Food Safety and Applied Nutrition conducts safety assessments of food additives, including food-contact substances such as polymeric and oligomeric materials that have the potential to migrate to food. Traditionally, little toxicity testing has been conducted on the low-molecular weight oligomeric fraction (< 1000 Da) of these food-contact substances. At lower exposures (≤ 150 µg/person/day), safety has been assessed based on the use of toxicity data on the monomeric components of these polymers as a sufficiently conservative approach for addressing the concern for genetic toxicity and carcinogenicity of the low-molecular weight oligomers (LMWOs). This paper discusses this assumption relative to the available data on these substances and their monomeric components in the context of exposures of ≤ 150 µg/person/day with emphasis on the evaluation of the potential genetic toxicity of these compounds. In most instances, data are available on either the monomers or the monomers' structural class to conservatively address the potential genetic toxicity of the LMWOs. Caveats to this generalization are also discussed. The assessment of LMWOs is important because they can be one of the primary migrants to food from a polymeric food-contact substance.

Affinity extraction combined with stable isotope dilution LC/MS for the determination of 5-methyltetrahydrofolate in human plasma.

The predominant circulating folate monoglutamate in human plasma (>90%), and thus the most significant folate for accurately diagnosing folate deficiency, is 5-methyltetrahydrofolic acid (5 MT). Folate deficiency is typically indicated when circulating folate levels are < or = 3 ng/mL. The quantitative determination of plasma folates in general, and of 5 MT in particular, is complicated by their naturally low levels (pg/mL to ng/mL), their instability, and their tendency to interconvert. Highly specific and sensitive analytical methods are needed to accurately quantify endogenous 5 MT in human plasma. A method that utilizes the specific high-affinity binding sites of bovine folate binding protein (FBP) and the selectivity and sensitivity of selected ion monitoring mode isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to quantify plasma 5 MT has been developed. The method is based on the solid-phase affinity extraction (SPAE) of 5 MT and its stable isotopically labeled analogue ([13C(5)]5 MT) from plasma (1 mL) using FBP immobilized to polymeric beads. The excess high-affinity binding sites on the affinity columns enable quantitative extraction of 5 MT from plasma under optimized sample pH conditions. Additionally, it is demonstrated that plasma proteins do not hinder the determination of 5 MT; therefore, protein precipitation is not required before the affinity extraction step. Detection and quantification of the extracted 5 MT is provided by positive-ion mode LC/MS in which the protonated molecular ions [M+H](+) of the analyte and the internal standard are monitored. The method shows linearity over three orders of magnitude (0.04-40 ng/mL) and has limits of detection and quantification of 0.04 and 0.4 ng/mL, respectively. Calibration curves obtained by spiking 5 MT into plasma exhibited good linearity between 0 and 25 ng/mL and both the plasma calibration standards and the plasma samples were stable for at least 48 h at room temperature. The recovery (average +/- % RSD) of 5 MT spiked into plasma from 5 to 25 ng/mL was 98.0% +/- 1.6% (n = 15). 5 MT levels determined by SPAE-LC/MS compared to "total folate" levels determined by radioassay and microbiological assay were discordant. Reasons for the discordancy are theorized, but it is clear that there exists an urgent need for clinical reference materials containing certified folate levels.

Solid-phase extraction-electrospray ionization mass spectrometry for the quantification of folate in human plasma or serum.

The measurement of 5-methyltetrahydrofolic acid (5 MT) blood levels is one of several factors used to diagnose folate deficiency in humans. 5 can be selectively purified from either human plasma or human serum via solid-phase extraction procedures and specifically detected and quantified in the extracts with liquid chromatography/isotope-dilution electrospray-ionization mass spectrometry. Two different, yet complementary, solid-phase extraction-liquid chromatography/mass spectrometry methods have been developed and applied to the quantification of 5 MT from such extracts. One method utilizes the high-affinity folate-binding protein from cow's milk coupled with multiple-reaction-monitoring-mode tandem mass spectrometry while the other method utilizes reversed-phase C(18) extraction followed by selected-ion-monitoring-mode mass spectrometry. The accuracy of each method is assessed through a comparative determination of 5 MT levels in homogenous plasma and serum pools. Additionally, each method is compared and evaluated against the "total folate" results provided by routine radioassay and microbiological assay determinations. On the basis of the experimental data presented in this report, it is suggested that both methods have the capacity to serve as potential reference methods for the quantification of circulating 5MT in plasma or serum.