Examination of Aggregate Formation upon Simultaneous Dissolution of Methacrylic Acid Copolymer LD Enteric Coating Agent, Pharmaceutical Additives, and Zwitterionic Ingredients.

We previously showed that adhesive aggregates were formed when levofloxacin hydrate tablets and lansoprazole orally disintegrating (OD) tablets were suspended in water in the clinical context. In this study, we have clarified the factors causing aggregate formation, focusing on the role of pharmaceutical additives and electrostatic interaction. Co-suspension of enteric-coated proton pump inhibitor (PPI) esomeprazole magnesium hydrate with levofloxacin resulted in aggregate formation, whereas the non-enteric-coated PPI vonoprazan fumarate did not. A comparison of pharmaceutical additive in the two PPIs highlighted polysorbate 80 and methacrylic acid copolymer LD as candidates causing aggregation. When these pharmaceutical additives were added to levofloxacin, only methacrylic acid copolymer LD induced aggregate formation. Since levofloxacin is zwitterionic, we examined another zwitterionic ingredient, ampicillin sodium, and found that it also formed aggregates with methacrylic acid copolymer LD, while benzylpenicillin sodium, which is not zwitterionic, did not form aggregates. When we next examined a series of zwitterionic quinolone antimicrobial drugs, we found that ofloxacin, which is highly soluble, formed aggregates with lansoprazole OD tablets, whereas poorly soluble quinolone antimicrobial drugs did not form aggregates. Further, although cefepime hydrochloride and cephalexin did not form aggregates with methacrylic acid copolymer LD in tap water, aggregates were formed when a suspension of cefepime hydrochloride or cephalexin with methacrylic acid copolymer LD was adjusted to pH 7.0. Our results indicate that electrostatic interaction between zwitterionic ingredients and methacrylic acid copolymer LD can result in aggregate formation under conditions where the drug and methacrylic acid copolymer LD are both sufficiently soluble.

[Determination of Antifungal Drugs in Fish and Livestock Products Using LC-MS/MS].

We developed an analytical method for determining 15 antifungal drugs, 2 antiparasitic drugs, and 3 veterinary drugs in fish and livestock products using LC-MS/MS. First, 50% ethanol was added to their products, and the mixture was homogenized to reduce drug degradation. Thereafter, 20 drugs were extracted from the pretreated sample mixture using acetonitrile. Cleanup was performed using an alumina-N SPE cartridge. Finally, chromatographic separation was performed using a fully porous octadecyl silanized silica column. The new method is applicable to fish in which the matrix hampers accurate analysis. It was validated on 8 fish and livestock products. Drug recovery rates ranged from 70.2 to 109.3%, RSDs of repeatability were <18.0%, and RSDs of within-laboratory reproducibility were <18.7%. It fulfills the Japanese guideline criteria. The limits of quantification were estimated as 3 ng/g.

[Determination of Cyromazine in Livestock Products by LC-MS/MS on an Anion-Cation Exchange Mode ODS Column].

Cyromazine in livestock products was determined using a validated LC-MS/MS method. There are three key points in our methods. First, the extraction was performed with two solutions, methanol and pH 3.0 McIlvaine buffer. The process was optimized for each type of sample. Secondly, cleanup was performed using a reversed-phase and strong cation exchange mixed-mode cartridge. The cartridge was washed with 0.14％ ammonium solution. Thirdly, the chromatographic separation was performed on an anion-cation exchange mode ODS column. There was no matrix effect on the extraction and determination for five livestock products. The quantification was carried out using an external standard calibration curve. This new method satisfies the Japanese guideline criteria. Recovery ranged from 77.2 to 92.1％, the relative standard deviation of repeatability （RSDr） was under 2.2％, and RSDwr was under 6.1％. Residual cyromazine was detected in raw milks and eggs.

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila.

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.

Quantification of staphylococcal enterotoxin type A in cow milk by using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

In this study, the staphylococcal enterotoxin type A (SEA) contaminant was quantified in cow milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin's presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 µg kg-1 conducted with two samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The relative standard deviations of repeatability were 13-14% and the relative standard deviations of within-laboratory reproducibility were 13-18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The quantification limit was estimated to be 10 µg kg-1.

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS.

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSDr) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSDWR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 µg kg-1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 µg kg-1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.

Identification and quantification of cereulide in cow's milk using liquid chromatography-tandem mass spectrometry.

In this study, the presence of cereulide in cow's milk was identified and quantified using our validated method with liquid chromatography-tandem mass spectrometry. Cereulide was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of cereulide. Besides the matrix effect (-4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 µg kg-1), using three samples daily on five different days based on the Japanese guidelines. This new method exhibited good accuracy ranging from 91% to 94%. The relative standard deviations of repeatability ranged from 2% to 5%, and the relative standard deviation of within-laboratory reproducibility ranged from 5% to 6%. These standard deviations satisfied the criteria for the Japanese validation guidelines. The limit of quantification (LOQ) was estimated to be 2 µg kg-1. On the product ion spectra at the LOQ level, the library match was satisfactory with a purity fit value of >70%. The method was applied to 14 raw milk and three milk samples pasteurised using the low-temperature, long-time process and collected in Tokyo. None of the samples was found to contain the target toxin.

Rapid Multi-Residue Analysis of Pesticides in Pulses by LC-MS/MS.

Rapid multi-residue analysis of pesticides in pulses was developed using LC-MS/MS. Pesticide residues in 5 g of homogenized pulses were extracted with 30 mL of acetonitrile and salted out with 4 g of anhydrous magnesium sulfate and 2 g of sodium chloride in the presence of citrate buffer in a disposable tube. The resulting residues were extracted with 30 mL of acetonitrile, and co-extractives were removed on a handmade four-layer column, consisting of a layer of Z-Sep/C18 (20 mg/50 mg) dry particles on top of a three-layer, custom-made (pre-packed) column (lower bed: 60 mg of PSA, middle bed: 30 mg of GC, and top bed: 60 mg of C18) packed in a 10 mm internal diameter polypropylene column (3 mL). The developed method showed good recoveries of pesticides in soybean, lentil, white kidney bean and garbanzo. According to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan, recovery tests were conducted in soybeans fortified with 107 kinds of pesticides at the levels of 0.01 and 0.1 µg/g, respectively. At each concentration 2 samples were extracted on 5 separate days. Pesticides in the test solution were determined by LC-MS/MS using scheduled MRM. As regards the trueness of this method for 107 pesticides in soybeans, 97 pesticides were in the range of 70-120% with satisfactory repeatability and within-run reproducibility. This new method is expected to be applicable for routine examination of pesticide residues in soybeans.

An infant with idiopathic orbital myositis poorly responsive to steroid therapy: a case report.

A 9-month-old girl developed subacute limited adduction of the left eye, presenting with blepharoptosis. An orbital magnetic resonance imaging (MRI) 2 months after the onset revealed swelling of the left lateral rectus muscle, with increased intensity on T2-weighted images with fat saturation, which was enhanced with gadolinium. She was diagnosed with idiopathic orbital myositis based on history, physical examination, and MRI findings. Swelling of the left lateral rectus muscle was partially reduced by pulse steroid therapy. This is the first reported case of an infant orbital pseudotumor with clinical and MRI findings consistent with subacute orbital myositis. We propose that a fibrotic change of the orbital muscle may occur during a subacute course and would be incompletely responsive to steroid therapy.

Increased levels of insulin-like growth factor binding protein-3 in hypertensive patients with carotid atherosclerosis.

BACKGROUND: It has been proven that the intima-media thickness (IMT) of the carotid artery increases in patients with essential hypertension. Serum levels of insulin-like growth factor-1 (IGF-1) increase in hypertensive patients with ventricular hypertrophy. However, the relationship between carotid atherosclerosis and serum levels of IGF-1 and its binding protein-3 (IGFBP-3) in patients with essential hypertension has not been established. METHODS: The carotid IMT, blood pressure (BP), serum lipid profiles, and serum IGF-1 and IGFBP-3 contents were determined in 54 hypertensive patients (19 with and 35 without carotid plaque) and 52 normotensive controls without plaque. RESULTS: Systolic, diastolic, and mean BPs and serum IGFBP-3 level were significantly higher in the hypertensive patients (with and without plaque) than in the normotensive controls. The IGFBP-3 level correlated with systolic BP (r = 0.204, P =.0354). Age, gender, body mass index, and serum levels of HDL cholesterol, triglycerides, lipoprotein(a), lipid peroxides, insulin, and fasting plasma glucose did not differ significantly among the three groups. Hypertensive patients with plaque, compared with those without plaque or the normotensive controls, had the highest values of carotid IMT, LDL cholesterol, IGF-1, and IGFBP-3. Multiple logistic regression analysis revealed that the IGFBP-3 level was associated with a ninefold (95% confidence interval 2.6-31) higher risk of carotid plaque formation compared with LDL cholesterol or IGF-1 levels. CONCLUSIONS: These results suggest that an increased level of IGFBP-3 may play a crucial role in the development of carotid atherosclerosis in hypertensive patients.

Small dense low-density lipoprotein and carotid atherosclerosis in relation to vascular dementia.

Vascular dementia (VaD) and Alzheimer's disease (AD) are the most common causes of dementia in the elderly. The aim of this study was to investigate carotid atherosclerosis, serum lipid profiles, and atherogenic hormone levels in nondiabetic Japanese men with VaD or AD. Carotid artery intima-media thickness (IMT) and plaque, serum lipid and lipoprotein profiles, including low-density lipoprotein (LDL) particle size, as well as insulin-like growth factor-I (IGF-I, somatomedin C) and testosterone levels, were determined in 34 patients with AD, 37 patients with VaD, and 63 healthy male controls. Age, body mass index, systolic and diastolic blood pressure, and fasting plasma glucose, hemoglobin A(1c) (HbA(1c)), triglyceride, high-density lipoprotein (HDL)-cholesterol, and apolipoproteins (apo) A-I, B, and E levels did not differ significantly among the 3 groups. However, the mean value of carotid IMT, the frequency of atherosclerotic plaque deposition, the serum levels of LDL-cholesterol, lipoprotein(a), and lipid peroxides, and the incidence of small dense LDL (particle diameter

[Studies on rapid multi-residue analysis of pesticides in agricultural products by LC-MS/MS].

Rapid multi-residue analysis of pesticides in agricultural products was studied by using LC-MS/MS. Pesticide residues in 10 g of homogenized agricultural products were extracted with 30 mL of acetonitrile and salted out with 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride in the presence of citrate salts for buffering in a disposable tube. Co-extractives were removed by use of our original triple layered column (C18/GC/PSA; 60/30/60 mg). According to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan, we conducted recovery tests in 5 kinds of agricultural products (brown rice, kiwi, cabbage, sweet potato and spinach) spiked with 60 pesticides at the level of 0.01 or 0.1 µg/g. Each concentration of pesticide spiked was extracted from 2 samples per day on 5 days. Pesticides in the test solution were determined by two types of LC-MS/MS using scheduled MRM. Using this method, 58 out of 60 pesticides satisfied the guideline criteria in brown rice, 59 in kiwi, 55 in cabbage, 55 in sweet potato and 56 in spinach. This method is applicable for routine examination of pesticide residues in agricultural products.