[Determination of Caffeine, Theobromine, and Theophylline in Chocolate Using LC-MS].

A simultaneous determination method for caffeine, theobromine, and theophylline in chocolate was developed. Three compounds were ultrasonically extracted twice (15 min at 50℃) in an acetonitrile-water (1 : 1, v/v). The extract was purified using Oasis HLB SPE cartridge, and the purified processed by LC-MS. The method exhibited recoveries of 97.4-100.2%, RSDs of repeatability of 1.0-2.8%, and RSDs of within-laboratory reproducibility of 2.0-7.9%. This method was simpler and more selective than existing methods, and was practical for caffeine, theobromine, and theophylline analysis in chocolate.

[A Rapid and Simple Method for Detection of Colchicum autumnale Using PCR].

Colchicum autumnale is a perennial, toxic plant that originated in Europe and North Africa. Although inedible, it is occasionally consumed accidentally because it resembles the edible Allium victorialis and other related species. This misidentification has led to episodes of food poisoning in Japan. However, determining the causative agent of a food poisoning outbreak by observing the sample visually or analyzing the chemical composition is challenging when dealing with small samples. Therefore, we developed a novel set of PCR primers that anneal to the internal transcribed spacer (ITS) region of C. autumnale ribosomal DNA, designed to detect the presence of C. autumnale in small samples. These primers successfully detected C. autumnale in all samples in which it was present, and did not give a positive PCR band in the 48 other distinct crop species tested, in which it was not present. Further, our method could amplify DNA from samples of C. autumnale that had been heat-treated and digested using artificial gastric fluids. Thus, this PCR strategy is highly specific and can be used to distinguish C. autumnale simply and rapidly from various other crops.

Quantification of amygdalin, prunasin, total cyanide and free cyanide in powdered loquat seeds.

Amygdalin, prunasin, total cyanide and free cyanide concentrations in 12 powdered loquat seeds were investigated. Loquat (Eriobotrya japonica) is a species of flowering plant in the family Rosaceae, and its fruit is quite popular in Japan. Amygdalin and prunasin were measured using LC-MS/MS. Total cyanide was measured by enzymatic treatment, steam distillation and colorimetric quantification using the pyridine-pyrazolone method. Free cyanide was measured without enzymatic treatment. The mean concentrations of amygdalin, prunasin, total cyanide and free cyanide in powdered loquat seeds were 5900, 760, 410 and 44 mg/kg, respectively. The range of each quantitative value was extensive. Seven out of twelve samples were at risk for exceeding the acute reference dose (ARfD) of cyanide.

[Survey of glycoalkaloids content in the various potatoes].

The content of potato glycoalkaloid (PGA) was investigated in 27 cultivars of raw potatoes and 31 potatoes in commercial foods with peel. The investigation of the 27 cultivars of potatoes showed different contents of glycoalkaloids. "May queen" and "Sherry" showed high contents of PGA (180 mg/kg and 320 mg/kg, respectively) among the raw potatoes of middle size (ca. 100 g). On the other hand, "Inca red" showed the lowest content of 21 mg/kg. Higher contents of PGA were found in smaller potatoes in this study. The content of PGA varied in the range of 48-350 mg/kg in the potatoes in commercial foods with peel.

[Example of examination of allergic substances (egg, milk) in commercial foods].

Allergic substances (eggs, milk) in commercial foods were measured by means of the notified ELISA methods using FASTKIT ELISA kit (N kit) and "Tokuteigenzairyo sokutei kit" (M kit). Some samples were also analyzed with the notified western blot method using "Tokuteigenzairyo western blot kit". In the methods for detection of eggs, proteins of fish egg and chicken muscle were examined using the ELISA and Western blotting methods to check for cross-reaction. Sujiko was false positive with the M kit. Difficulties were also encountered in the notified ELISA and Western blotting methods with raw chicken. In foods labeled as containing eggs, it was difficult to detected in the heart-processed food using both notified methods. In the method for detection of milk, foods containing thickening polysaccharides, showed cross-reaction with the N kit. However, this cross-reaction was eliminated when an improved N kit was used. Foods labeled as containing milk did not present any difficulty.

[Survey of histamine content in seafood on the market].

Food poisoning involving histamine has occurred almost every year for 20 years in Tokyo, and is usually due to ingestion of fish with lean meat, such as sardine, mackerel, horse mackerel and so on. Therefore, we were investigated the levels of histamine and 4 non-volatile amines (tyramine, putrescine, cadaverine, spermidine) in 637 samples on the market. The water activity of samples in which histamine was detected at 5 mg/100 g and over was examined. Histamine, tyramine, putrescine, cadaverine and spermidine were detected in 66, 43, 26, 64 and 5 samples, and the detection ranges were 5-340, 5-51, 5-42, 5-180 and 5-8 mg/100 g, respectively. Most of the samples in which histamine was detected were semi-dried round and split sardine. Water activity of 24 samples of semi-dried round and split sardine in which histamine was detected was in the range of 0.68-0.96.

[Contents and its change during storage of alpha-solanine and alpha-chaconine in potatoes].

Contents of alpha-solanine and alpha-chaconine in native species of potato (May Queen, Danshaku and Waseshiro), and in species (Jagakids Red '90 (Red) and Jagakids Purple '90 (Purple)) on the market, and their change during storage at room temparature were investigated. alpha-Solanine and alpha-chaconine were extracted from potatoes with methanol, cleaned up by using a Sep-Pak Plus C18 cartridge, and then subjected to HPLC. The recoveries of alpha-solanine and alpha-chaconine from potatoes were both more than 96%, and the quantitation limits were both 2 microg/g. alpha-Solanine and alpha-chaconine were detected in periderm in all samples at the levels of 260-320 microg/g in May Queen,190-240 microg/g in Danshaku, 43-63 microg/g in Waseshiro, 140-200 microg/g in Red and 84-130 microg/g in Purple, respectively. alpha-Solanine and alpha-chaconine were detected in the cortex in all samples of May Queen and Danshaku at the levels of 2.7-12 microg/g and 5.8-31 microg/g, respectively. Contents of alpha-solanine and alpha-chaconine in the cortex of May Queen and Danshaku were less than 10% of those in the periderm. When potatoes were stored for 90 days at room temparature in a dark place, no marked change in the contents of alpha-solanine and alpha-chaconine was observed in any of the potato samples.

[Structural analyses of barbaloin-related compounds in aloe drinks].

Four kinds of barbaloin (BA)-related compounds (A, B, C, D) in aloe drinks were isolated by using preparative HPLC. LC/MS analyses of these compounds showed the identical quasimolecular ion peak at m/z 833 [M-H]-. The chemical structures were mainly determined by NMR, including 1H-1H two-dimensional correlation spectroscopy (COSY), nuclear Overhauser and exchange spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple-bond connectivity (HMBC) experiments. They were identified as BA-related compounds A (10R, 10"S), B (10S, 10"S), C (10S, 10"R), and D (10R, 10"R) coupled through a C-10 to C-7' linkage, and newly found in nature. These results suggested that BA is converted to dimers during storage of aloe drinks.

[Contents of barbaloin-related compounds in aloe drinks and their change during storage].

The contents of barbaloin (BA), isoBA, aloin-dimers A, B, C, D and aloe-emodin (AE) in aloe drinks were investigated. BA and isoBA were detected in 30 of the 31 samples at the levels of 120-570 micrograms/mL and 120-580 micrograms/mL, respectively. Aloin-dimers A, B, C and D were detected in 8 of the 31 samples at the levels of 12-38 micrograms/mL, 13-39 micrograms/mL, 11-36 micrograms/mL and 16-69 micrograms/mL, respectively. AE was detected in all of the 31 samples at the levels of 0.03-1.3 micrograms/mL. When aloe drinks were stored for 4 weeks at 5 degrees C after opening the bottle, decrease of BA and isoBA, and increase of AE and aloin-dimers A, B, C and D were observed in most cases. However, in a few aloe drinks, all of BA, isoBA, aloin-dimers A, B, C, D and AE decreased. In these drinks, the existence of aloin-trimer was elucidated by LC/MS analyses. These data suggested that BA in aloe drinks is converted to the dimer and then to the trimer during storage.