Vitamin B₁₂ was obtained from Sigma (St Louis, Missouri, USA). A vitamin B₁₂ assay medium based on Lactobacillus delbrueckii subspecies lactis (formerly L. leichmannii) ATCC 7830 was obtained from Nissui (Tokyo, Japan). Silica gel 60 thin layer chromatography (TLC) aluminum sheets were obtained from Merck (Darmstadt, Germany). Canned escargots (boiled plain) were purchased from local markets in Japan.

After broth of canned escargots (boiled plain) was removed, all solid content (escargot whole body) was homogenized using a mixer (TML160; Tescom & Co., Ltd., Tokyo, Japan). An aliquot (2.0 g) of the homogenate was used as the sample. The vitamin B₁₂ compounds were extracted from the sample by boiling at pH 4.5 and then assayed using a microbiological method based on L. delbrueckii ATCC 7830, according to a previously described method [4] . L. delbrueckii ATCC 7830 can utilize deoxyribosides and deoxyribonucleotides (known to be an alkali-resistant factor) as well as vitamin B₁₂. Thus, the correct vitamin B₁₂ values were calculated by subtracting the values for the alkali-resistant factor from the total vitamin B₁₂ values.

A bioautography assay to detect corrinoid compounds was performed as previously described [6] . The vitamin B₁₂ extract (20 mL) prepared as described above was partially purified and concentrated using a Sep-Pak Plus^®C18 cartridge (Waters Corp., Milford, MA) that was washed with 5 mL of 75% (v/v) ethanol and equilibrated with 5 mL of distilled water. The C18 cartridge was washed with 5 mL of distilled water and vitamin B₁₂ compounds were eluted using 2 mL of 75% (v/v) ethanol. The eluate was evaporated in a centrifugal concentrator (Integrated Speed Vac^® System ISS110; Savant Instruments Inc., NY, USA), and the residual fraction was dissolved in 1.0 mL of distilled water. Concentrated extracts (1 μL) and vitamin B₁₂ and pseudovitamin B₁₂ (each 0.1 mg/L) were spotted onto the silica gel 60 TLC plates and developed in the dark using 2-propanol/ NH₄OH (28%)/water (7:1:2 v/v) at room temperature (25˚C). The TLC plate was dried and overlaid with agar-containing basal medium and precultured E. coli 215, followed by incubation at 30˚C for 20 h. The gel plate was subsequently sprayed with methanol solution containing 2,3,5-triphenyltetrazolium salt, and vitamin B₁₂ compounds were visualized as red, which indicated E. coli growth.

Each vitamin B₁₂ extract (40 mL) was partially purified and concentrated using a Sep-Pak^® Plus C18 cartridge (Waters Corp.) as described above. The eluate was evaporated to dryness under reduced pressure and the residual fraction was dissolved in 3 mL distilled water and centrifuged at 10,000 × g for 10 min to remove insoluble material. The supernatant fraction was loaded onto an immunoaffinity column [EASI-EXTRACT^®B₁₂ Immunoaffinity Column (P80) R-Biopharm AG, Darmstadt, Germany], and vitamin B₁₂ compounds were purified according to the manufacturer’s protocol. The vitamin B₁₂ compounds were purified by passage through the immunoaffinity column, dissolved in 0.1% (v/v) acetic acid, and filtered through a Nanosep MF centrifuge device (0.4 μm; Pall Corp., Tokyo, Japan) to remove small particles. An aliquot (2 μL) of the filtrate was analyzed using a LC/MS IT-TOF (ion trap-time-of-flight) system coupled to an Ultra-Fast LC system (Shimadzu, Kyoto, Japan). Each purified corrinoid was injected into an Inert-Sustain column (3 μm, 2.0 × 100 mm; GL Science, Tokyo, Japan) and equilibrated with 100% solvent A [0.1% (v/v) acetic acid] and 0% solvent B (100% methanol) at 40˚C. Corrinoids were eluted using a linear gradient of methanol (15% solvent B for 0 - 5 min, 15% - 90% solvent B for 5 - 11 min, and 90% - 15% solvent B for 11 - 15 min). The flow rate was 0.2 mL/min. ESI conditions were determined by injecting the corrinoids into the MS detector, thereby identifying the optimum parameters for detecting parent and daughter ions of vitamin B₁₂ compounds. The ESI-MS system was operated in the positive ion mode, and argon was used as the collision gas. Vitamin B₁₂ (m/z 678.2914), factor IIIm or methoxymensimidazolyl cyanocobamide (m/z 679.7834), and factor S or 2-methylmercaptoadenyl cyanocobamide (m/z 695.7657) and as [M + 2H]²⁺ were confirmed by comparing the observed molecular ions and retention times.

We analyzed the vitamin B₁₂ contents of canned escargots (boiled plain) that are available in Japan using the L. delbrueckii ATCC 7830 microbiological assay method (Table 1). Vitamin B₁₂ contents of canned escargots (boiled plain) were varied from approximately 0.8 μg/100g weight to approximately 5.5 μg/100g weight. Mean values (2.2 μg/100g weight) were approximately 3.6-times greater than the value (0.6 μg/100g weight) of the Japanese food composition database [7] , but is very lower than values (17.4 - 39.4 μg/100g weight) of canned clams (boiled plain) [8] .

The corrinoids observed in all escargot samples were analyzed using an E. coli 215 bioautogram after separation by silica gel 60 TLC. The corrinoids observed in all escargot samples produced clear spots which had the identical Rf value of authentic vitamin B₁₂, although sample A and B showed another sport, which was not identical to that of pseudovitamin B₁₂ (Figure 1).

To evaluate escargot vitamin B₁₂ compounds, each vitamin B₁₂ extract was purified using a vitamin B₁₂ immunoaffinity column and analyzed by LC/ESI-MS/MS. Authentic vitamin B₁₂ was eluted as peak with a retention

Total vitamin B₁₂ compounds were extracted from an aliquot (2.0 g) of each sample homogenate by boiling at pH 4.5 in the presence of 4.0 × 10⁻⁴% KCN and assayed using the Lactobacillus delbrueckii ATCC 7830 microbiological assay. The vitamin B₁₂ assay was performed in triplicate.

time of 7.50 min (Figure 2(a)). Mass spectrum of authentic B₁₂ indicated that a doubly charged ion with an m/z of 678.2883 [M + 2H]²⁺ was prominent (Figure 2(b)). The exact mass calculated from its formula (C₆₃H₈₈CoN₁₄O₁₄P) was 1354.5674, and the isotope distribution data showed that vitamin B₁₂ was the major doubly charged ion under the LC/ESI-MS conditions used in our assay. The MS/MS spectrum of authentic vitamin B₁₂ indicated that the dominant ion at m/z 359.0984 was attributable to the nucleotide moiety. The vitamin B₁₂ compounds purified from escargots were eluted as three ion peaks with m/z 679.7834, m/z 678.2914, and m/z 695.7657 at retention times of 7.35 min, 7.50 min, and 7.65 min, respectively (Figure 3(a)). The mass spectrum of the ion peak

with m/z 678.2914 at a retention time of 7.50 min showed that the doubly charged ion was formed at m/z 678.2876 (Figure 3(b)). The MS/MS spectrum of the compound was identical to that of vitamin B₁₂ (Figure 3(c)). The mass spectra of the ion peaks with retention times of 7.35 min and 7.65 min showed that the doubly charged ions were formed at m/z 679.7817 and 695.7633, respectively (Figure 3(d) and Figure 3(f)). The MS/MS spectra of the ion peaks of m/z 679.7817 and 695.7633 indicated that the dominant ions at m/z 362.0846 and m/z 394.0537, respectively, were attributable to each nucleotide moiety of these compounds; these spectral data coincided with the masses of nucleotide moieties of factor IIIm or methoxybenzimidazolyl cyanocobamide (C₆₂H₈₆CoN₁₄O₁₅P, 1356.5467) and factor S or 2-methylmercaptoadenyl cyanocobamide (C₆₀H₈₅CoN₁₇O₁₄PS, 1389.5252) (Figure 4). The results indicated that canned escargots (boiled plain) contained vitamin B₁₂ and other two inactive corrinoid compounds, which are identified as factor IIIm and factor S. The similar results were obtained in the remaining samples B, C, D, and E. Relative contents of factor IIIm (34.4% ± 8.6%) and factor S (25.8% ± 6.9%) against vitamin B₁₂ (100%) were shown in these samples from calculating height of respective three peaks at 360 nm.

Vitamin B₁₂ content (2.2 ± 1.9 μg/100g weight) of canned escargots (boiled plain) was very lower than values (17.4 - 39.4 μg/100g weight) of canned clams (boiled plain). Moreover, escargots contained two inactive corrinoid compounds, which were identified factor IIIm and factor S, as well as vitamin B₁₂. It remains unclear why these inactive corrinoids were present in escargots. Further detailed biochemical studies are required to elucidate the origins of these inactive corrinoid compounds. Escargots would be not suitable for use as a source of vitamin B₁₂.