[Simultaneous Analysis of 7 Antiviral Agents in Chicken Tissues and Processed Products by LC-MS/MS].

Since amantadine, rimantadine, arbidol, laninamvir, oseltamivir, peramivir, and zanamivir may be used as antiviral agents to treat avian influenza, we herein developed a simultaneous assay using LC-MS/MS. This method was applied to chicken products (including yakitori (grilled chicken), fried chicken, chicken steak, and boiled eggs) as well as chicken tissues (muscle, fat, the liver, gizzards, and heart) and eggs.Samples were extracted with methanol-water (9 : 1), purified by a tandem column with an InertSep® MAX cartridge (upper part) and InertSep® MCX cartridge (lower part), and then measured by LC-MS/MS. The sample matrix had no effect on the identification of compounds. Chromatographic separation was performed on a ZIC-HILIC column using a mobile phase of 1% acetic acid solution and 1% acetic acid solution in acetonitrile, resulting in complete separation and other obstructive peaks from the sample matrices. An external solvent calibration curve was used for quantification.The application of the method to 6 samples of chicken tissues and eggs achieved good results of between 77.9 and 97.5% for trueness and between 1.7 and 9.2% for concurrent accuracy. The method was also applied to 9 samples of processed products, including grilled chicken and fried chicken, and achieved good results with true percentages ranging between 72.6 and 99.2% and concurrent accuracies between 3.0 and 11.2%. Therefore, the developed method may also be applied to processed products.The limit of quantification (LOQ) of the developed method was 0.01 mg/kg.The method was then applied to 42 types of commercial processed products, including yakitori, fried chicken, steamed chicken, chicken steak, and boiled eggs, and no antiviral agents were detected.Collectively, the present results confirmed that the method developed herein is applicable to not only chicken tissues, but also their processed products.

Evaluation of the accuracy of urine analyzers in dogs and cats.

The accuracy of urine analyzers used for dogs and cats has remained uncertain. This study examines the agreement between results of urine analysis obtained using two devices marketed for animals and for humans and the results of quantitative biochemical analysis. The degrees of concordance for bilirubin and ketones in the same category were ~80%, but for pH these were only ~60% in dogs and cats. Degrees of concordance for protein and the UP/C ratio clearly differed between the devices for animals and humans. We found that values for bilirubin and ketones obtained using urine analyzers may be reliable, but pH is unlikely to be accurate enough to be clinically useful for dogs and cats.

[Determination of Zilpaterol in Livestock Products by LC-MS/MS].

An analytical method for the determination of zilpaterol in livestock products was developed. The sample was stirred with n-hexane and n-hexane saturated acetonitrile, and zilpaterol in the sample was extracted with acetonitrile. The extract was cleaned up on a ODS cartridge column (1 g) and SCX cartridge column (500 mg). The LC separation was carried out using an Inertsil ODS-4 column and linear gradient elution with 0.1%formic acid and acetonitrile containing 0.1% formic acid as mobile phase. Detection of MS was carried out positive ion electrospray ionization mode. Average recoveries (n=5) of zilpaterol from 6 kinds of livestock products fortified at the MRLs (0.01 mg/kg) were 87.0-99.4%, and the relative standard deviations were 2.4-6.3%. The limits of quantitation were 0.01 mg/kg.

Determination of Diniconazole in Agricultural Products, Livestock and Marine Products by LC-MS/MS.

An LC-MS/MS method for the determination of diniconazole in agricultural products, livestock and marine products was developed. Diniconazole in agricultural products was extracted with acetone. The extract was concentrated and partitioned with n-hexane and 10% sodium chloride solution. Agricultural products such as grains and beans were defatted using n-hexane-acetonitrile. Livestock and marine products were extracted with a mixture of acetone and n-hexane, and the organic layer was evaporated to dryness. The residue was defatted using n-hexane-acetonitrile. Cleanup was carried out using a Florisil cartridge column and a graphitized carbon cartridge column for these samples. The LC separation was carried out on an Inertsil ODS-3 column with a linear gradient of 0.1% formic acid and acetonitrile containing 0.1% formic acid. MS was carried out in the positive ion electrospray ionization mode. The calibration curve was linear between 0.00125 to 0.00750 mg/L. Average recoveries (n=5) of diniconazole from 16 kinds of agricultural products, livestock and marine products fortified at the MRLs (0.01 ppm) were 88.3-108%, and the relative standard deviations were 0.5-5.1%. The limits of quantitation were 0.01 mg/kg.

[Prostate cancer in a relatively young adult: a case report].

We report a case of prostate cancer in a 45-year-old. The patient was found to have an elevated prostate specific antigen (PSA) level at a general health check and was referred to our division. Serum level of PSA was 6.9 ng/ml. Digital rectal examination and magnetic resonance imaging revealed a small nodule on the right lobe. Transperineal needle biopsy of the prostate revealed adenocarcinoma at the bilateral lobe (Gleason score 3+4). Computed tomography and a bone scan showed no evidence of metastasis. The patient was diagnosed as cT2b, NO, MO, and underwent a radical prostatectomy. Patients younger than 45 with prostate cancer are rare, although the incidence of prostate cancer is increasing. Here, 25 cases previously reported in Japan in addition to the present case, are reviewed.

[A case of synchronous ipsilateral renal cell carcinoma and renal pelvic urothelial carcinoma (collision tumor): a case report].

A 54-year-old woman consulted our hospital with the complaint of microscopic hematuria and proteinuria. Abdominal computed tomographic scan revealed a tumor mass on the lower pole of the left kidney. Left radical nephrectomy was performed under the diagnosis of renal cell carcinoma. The pathological diagnosis was a collision tumor consisting of renal cell carcinoma (clear cell type, G3, INFalpha, pT1b, v -) and urothelial carcinoma of the renal pelvic (G3 >> G2, pT3, ly + , v +). Postoperative chemotherapy was not given. Convalescence was uneventful and one year after the operation she is alive with no recurrence or metastasis.

[A case of emphysematous cystitis: a case report].

A 86-year-old woman presented with asymptomatic gross hematuria,and visited our hospital, and a vesicorectal fistula was suspected from a computed tomographic (CT) scan. She had a lower abdominal mass and urinary retention on arrival. Pclvic CT demonstrated intramural gas in the urinary bladder,which suggested a diagnosis of emphysematous cystitis. Cystoscopy demonstrated reddish mucosa and gas within the bladder wall. The gross hematuria was improved and the intramural gas disappeared on a CT scan after urinary drainage and antibiotic therapy. Although emphysematous cystitis is almost always cured with conservative therapy, an accurate diagonosis and prompt treatment are required because rarely emphysematous cystitis can result in rupture of the urinary bladder and lead to septic shock.

[Prostatic duct adenocarcinoma with pagetoid spread on the glans penis: a case report].

A 75-year-old male visited our division with asymptomatic erythema on the glans penis which he first noticed six months earlier. The patient underwent total cystoprostatectomy under the diagnosis of urothelial carcinoma of the urinary bladder four years earlier. At the time, the prostatectomy specimen incidentally revealed a prostatic acinar adenocarcinoma at the bilateral peripheral zone. A skin biopsy of the erythema revealed intraepithelial Paget's cells, and the patient underwent total penectomy under the diagnosis of extramammary Paget's disease. Histopathological examination revealed continuous intraepithelial Paget's cells from the glans penis to the urethral navicular fossa, and a ductal carcinoma was detected beneath the urethral mucosa to the excisional margin. Because the Paget's cells expressed cytokeratin 20, the tumor was diagnosed as Pagetoid spread rather than Paget's disease. Re-examination of the previous prostatectomy specimen revealed prostatic duct adenocarcinoma with prostatic acinar adenocarcinoma. Therefore, the final diagnosis was prostatic duct adenocarcinoma with Pagetoid spread to the glans penis. Follow up at nine months revealed neither local recurrence, nor distant metastases, although no adjuvant therapy has been given.

[Determination of vitamin K in aojiru (green juice) products by HPLC].

A survey of vitamin K contents was carried out on 41 aojiru products and 10 vegetable juice products that were purchased from local markets. Aojiru is a health food made from green vegetables such as kale, ashitaba, mulberry leaf, barley grass and the like. The products are usually provided in various forms, such as frozen, powder and tablet. Vitamin K in samples was extracted with n-hexane, and separated on a C18 column with methanol-ethanol (95 : 5). After separation, vitamin K was converted to the hydroquinone form on a reduction column and determined with a fluorescence detector at lambdaex 240 nm and lambdaem 430 nm. The contents of vitamin K1 (phylloquinone) in frozen samples (n = 8), powder samples (n = 26) and tablet samples (n=7) were 90-190, 410-3,300, and 640-3,100 microg/100 g, respectively, and that in vegetable juice (n= 10) was 1-12 microg/100 g. Vitamin K2 (menaquinone) was not detected. The daily intake of vitamin K from aojiru products was estimated to be 99-380, 20-250 and 27-210 microg/day for frozen, powder and tablet types, respectively. These results suggest that patients prescribed warfarin should take care about their intake of vitamin K from aojiru products.

Determination of magnesium stearate in capsule- or tablet-type supplements.

A simple method for the determination of magnesium stearate in capsule- or tablet-type supplements was developed. Free stearic acid in the sample was removed by extraction with tetrahydrofuran. The remaining stearate was converted to stearic acid by reaction with a cation-exchange resin. The resulting stearic acid was determined by gas chromatography with a polar column. Esters of stearic acid were not converted to stearic acid and would not cause a positive error in the amount of stearate. The amount of magnesium stearate was calculated based on the stearic acid concentration thus obtained. Magnesium stearate levels in 5 out of 25 supplements exceeded 2500 microg/g, which indicated the possible admixture of magnesium stearate.

[Oncocytoma associated with acquired cystic disease of kidney (ACDK): a case report].

A case of renal oncocytoma associated with acquired cystic disease of kidney (ACDK) in a 56-year-old man is reported. He had received hemodialysis for 15 years because of chronic renal failure. Computed tomography (CT) was performed because of distention in the upper abdomen, revealing a right renal tumor. He underwent laparoscopic right nephrectomy, and was diagnosed with renal oncocytoma. There have been reported 8 cases of renal oncocytoma in hemodialysis patients, and our case was the third one associated with ACDK in the literature.

[A case report of bladder hernia associated with prostate cancer].

We report a case of bladder hernia. A 68-year-old man was admitted to our hospital for the management of prostate cancer. An egg-sized soft mass was palpated at his right inguinal region. Magnetic resonance imaging and cystography revealed that the mass was a bladder hernia. During radical prostatectomy, we had to resect the bladder hernia for safe regional lymphadenectomy. This hernia was the extraperitoneal type. The stage of prostate cancer was pT3b N0 M0. This is the third reported case of inguinal bladder hernia associated with prostate cancer in Japan.

[Spontaneous rupture of renal angiomyolipoma: a case report].

A 55-year-old woman presented with sudden right lower abdominal pain. Computed tomography demonstrated retroperitoneal hematoma associated with minus-density area, diagnosed as spontaneous rupture of angiomyolipoma. Super-selective transarterial embolization was performed, but anemia and right abdominal pain became worse in spite of conservative therapy including transfusion, indicating re-rupture of the renal tumor. Although partial nephrectomy was planned, right nephrectomy was finally performed because of massive intraoperative bleeding. Accurate diagnosis and prompt treatment are required when life-threatening rupture of renal tumor is suspected.

[Determination of 4-hexylresorcinol residues in prawns and crabs].

A method for the determination of 4-hexylresorcinol residues in prawn and crab meat by HPLC was developed. 4-Hexylresorcinol in prawn and crab meats was extracted with methanol using a homogenizer. The extract was diluted 4 times with water, and the diluted solution was passed through a C18 cartridge. The cartridge was washed with water and methanol-water (4 : 6), and then 4-hexylresorcinol was eluted with acetonitrile-0.1% phosphoric acid (55 : 45). The eluate was separated on a Capcell Pak C18 MG column with a mobile phase of acetonitrile-0.1% phosphoric acid (6 : 4) and 4-hexylresorcinol was determined with a UV detector (210 nm). Recoveries of 4-hexylresorcinol from commercial prawn and crab meats spiked at 1.0 and 10 microg/g were 82.4-92.2 and 88.9-91.8%, respectively. The determination limit of 4-hexylresorcinol was 1.0 microg/g in the samples.

[Simultaneous determination of stevioside, rebaudioside A and glycyrrhizic acid in foods by HPLC].

A method for the simultaneous determination of stevioside (Stev), rebaudioside A (RebA) and glycyrrhizic acid (GA) in foods was developed. These sweeteners were extracted from foods, except for dried fishes and shellfishes, by dialysis against Tris-HCl buffer (pH 9.0). Dried fishes and shellfishes were extracted with Tris-HCl buffer--methanol (2:8). The extracts were cleaned up with an Oasis MAX cartridge. The cartridge was washed with 0.05 mol/L sodium acetate (pH 4.0)--methanol (19:1), and the three sweeteners were eluted with 0.1 mol/L phosphoric acid--acetonitrile (1:1). Stev, RebA and GA in the eluate were chromatographed on a Develosil RPAQUEOUS-AR-5 (4.6 mm i.d. x 250 mm) column with 0.02 mol/L phosphoric acid-acetonitrile--methanol (90:55:5) as a mobile phase and monitored at 210 nm for Stev and RebA, and at 254 nm for GA. The recoveries of Stev, RebA and GA from 8 kinds of foods spiked at the level of 0.1 g/kg were 81.7-101%, 81.5-100% and 78.6-95.0%, respectively. The determination limits were 0.01 g/kg in samples.

[Enantiomeric determination of ephedrine derivatives in unregulated drugs using capillary electrophoresis].

Capillary electrophoresis (CE) was applied to enantiomeric separation of chiral ephedrine derivatives (d/l-ephedrine, d/l-methylephedrine, d/l-pseudoephedrine, and d/l-norephedrine) in unregulated drug products. Unregulated drugs, referred to as dietary supplements in U.S.A., have been used legally as tonic agents, but illegal substances such as ephedrine were often detected. Baseline separation of all enantiomers of ephedrine derivatives was achieved using an electrophoretic solution containing heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DM-CD) as a chiral selector. The optimal conditions were established to be: capillary column of fused silica (50 microm i.d. x 56 cm); running buffer of 20 mM DM-CD with 50 mM potassium dihydrogenphosphate background electrolyte, pH 2.6; capillary temperature of 20 degrees C; applied voltage of 30 kV; on-column detection at 195 nm; and injection pressure of 50 mbar x 3 s. Under these conditions, all four pairs of enantiomers were sufficiently resolved, and eight peaks were observed with resolution factors of greater than 1.5. The calibration curves of all enantiomers showed good linearity over the concentration range of 2.5-10 microg/ml (r =0.999). The present method was used in a survey of marketed products. The resultant chiral contents were reported and the analytical data were also compared with those from HPLC. This method is useful in the simple and rapid analysis of ephedrine derivatives in marketed products.

Environmental fate of bisphenol A and its biological metabolites in river water and their xeno-estrogenic activity.

Monitoring of bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] and its biological metabolites [4,4'-dihydroxy-alphamethylstilbene (DHMS), 2,2-bis(4-hydroxyphenyl)-1-propanol (BPA-OH), 2,2-bis(4-hydroxyphenyl)propanoic acid (BPA-COOH), and 2-(3,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)propane (3-OH-BPA)] in river waters was performed by solid-phase extraction and GC/MS determination. The concentrations of BPA, BPA-COOH, BPA-OH, and 3-OH-BPA in the river water ranged from 2 to 230 (8.8 x 10(-12) to 1.0 x 10(-9) M), from 5 to 75 (1.9 x 10(-11) to 2.9 x 10(-10) M), from 3 to 16 (1.2 x 10(-11) to 6.6 x 10(-11) M), and from 3 to 11 (1.2 x 10(-11) to 4.5 x 10(-11) M) ng L(-1), respectively. DHMS, an intermediate in the main degradation pathway of BPA, was not detected in any water sample. Under the aerobic conditions in the river water, BPA disappeared within 8 d of incubation, but BPA-COOH, BPA-OH, and tetraol remained in the supernatant after 14 d of incubation. For the xeno-estrogenic activity of BPA and the metabolites, their ability to bind to recombinant human estrogen receptor alpha in competition with fluorescence-labeled 17beta-estradiol was measured. Fifty percent inhibitory concentrations (IC50) of BPA, DHMS, 3-OH-BPA, and BPA-OH were approximately 1 x 10(-5), 1 x 10(-6), 3 x 10(-5), and 1 x 10(-2) M, respectively. In human cultured MCF-7 breast cancer cells, BPA increased cell numbers in a dose-dependent manner at concentrations from 10(-7) to 10(-5) M. For the BPA metabolites, DOHMS, 3-OH-BPA, and BPA-COOH caused the cells proliferation at concentrations from 10(-9) to 10(-6), from 10(-7) to 10(-6), and from 10(-5) to 10(-4) M, respectively. BPA-OH did not cause MCF-7 cells proliferation. These results indicate that BPA is mainly metabolized through oxidative rearrangement by bacteria in the river water, and intermediate bisphenols via minor metabolic pathways exist in river water. The presence of the bisphenols having the xeno-estrogenic effect suggests the necessity of monitoring those in river water, in the effluent waters from sewage plants, or in landfill leachate.

[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.

[Multiresidue analysis of pesticides in agricultural products by GC/MS, GC-ECD and GC-FTD using acetonitrile extraction and clean-up with mini-columns].

An analytical method was developed for determination of organochlorine, pyrethroid and nitrogen-containing pesticide residues in agricultural products by GC/MS, GC-ECD and GC-FTD. Pesticides were extracted with acetonitrile, and the organic layer was cleaned up on C18 and ENVI-Carb/LC-NH2 cartridges. Pesticides were determined by GC/MS, GC-ECD and GC-FTD. Detection limits of pesticides by GC/MS and GC were below 1-30 ng/g and 1-3 ng/g, respectively. Recoveries of 27 out of 53 pesticides determined by GC/MS and 47 out of 53 determined by GC were 70-120%. This method was useful for the multiresidue analysis of pesticides in agricultural products. Pesticide residues in 81 domestic crops were investigated from April 2001 to March 2002. Residues of 31 pesticides were detected from 21 crops.