The combination of methotrexate (MTX) and non-steroidal anti-inflammatory drugs (NSAIDs), which is frequently used for rheumatoid arthritis (RA) treatment, courses of adverse events. To prevent these, simultaneous monitoring of these compounds is available. Therefore, we developed a new method for MTX and NSAIDs such as loxoprofen (LP), meloxicam (MX), lornoxicam (LX), diclofenac (DF) and celecoxib (CX) determination using HPLC with simple pretreatment of human serum sample. The separation of MTX and 5 NSAIDs was performed on C6-Phenyl column with gradient elution using 10 mmol/L ammonium acetate aqueous solution and methanol, and achieved within 25 min of analytical runtime. Calibration curves using standards showed good linearity (r2>0.9998) in the range of 0.02-500 pmol/10 μL injection for MTX, 10-500 pmol for LP, 0.2-500 pmol for MX and DF, 0.05-500 pmol for LX and 2-500 pmol for CX. The detection limits of the proposed method were at least less than 96.0 fmol, and repeatability was less than 4.62 RSD%. In addition, acceptable precision of less than 10.19 RSD% and recovery of more than 54.4% of the method were obtained because the peaks of MTX and NSAIDs could be well-separated from those of interferings in serum. Therefore, the proposed method might be useful to avoid occurring adverse events, and confirm the curative effect of MTX and NSAIDs.
Carbon nanodots (CNDs) prepared from glutamic acid or glutathione in an electric furnace were characterized by capillary electrophoresis. Two major peaks were detected in the electropherograms by capillary zone electrophoresis, corresponding to anionic and less-charged CNDs. The effective electrophoretic mobility of the anionic CND formed from glutamic acid was almost identical over neutral to weakly alkaline pH range, and the CND would not contain significant amount of amino group. On the other hand, the effective electrophoretic mobility tended to decrease with decreasing pH at weakly acidic pH conditions, suggesting the functional groups of carboxylate moiety on the anionic CNDs. Dodecyl sulfate ion was added in the separation buffer to give anionic charge to the less-charged CND by adsorption. However, the anionic charge induced was little, and the dodecyl sulfate ion was not likely adsorbed on the less-charged CND and the CND would be hydrophilic.
Separation of flavanonol, phenylcoumaran and flavonolignans in Silybum marianum was examined using high-speed countercurrent chromatography (HSCCC). In order to prepare analytical standards, a flavanonol of aromadendrin (87.4% purity, 7.8 mg), three phenylcoumarans of jatrointelignan D (84.5% purity, 13.2 mg), dehydrodiconiferyl alcohol (76.1% purity, 1.4 mg) and dihydrodehydrodiconiferyl alcohol (93.0% purity, 5.8 mg), and three flavonolignans of silybin (88.8% purity, 35.6 mg), silydianin (99.3% purity, 25.1 mg) and silychristin (96.9% purity, 12.6 mg) were separated from the seeds of S. marianum using common column chromatography and ODS-HPLC, and identified by 1H and 13C NMR spectra. Then, HSCCC with the hexane/ethyl acetate/methanol/water (3 : 7 : 4 : 6, v/v) system was applied to the separation of aromadendrin, jatrointelignan D, silydianin and silybin. In this separation, it was revealed that silybin and silydianin were successfully separated from each other. The present HSCCC system was directly applied to the ethyl acetate extract and resulted in the separation of silybin. The overall results suggested that HSCCC is useful for the separation of bioactive compounds in S. marianum.
A gas chromatographic micro-packed column prepared with titanium dioxide (TiO2) particles was developed. This study reports the fundamental retention behavior of the column for several organic and inorganic gaseous compounds, and quantitatively analyzes the high-temperature degradation behavior of the injected organic compounds. The anatase TiO2 particles were prepared by hydrolysis of titanium (IV) isopropoxide. The particle size was classified as 150 – 180 μm. The micro-packed column was prepared by packing the classified TiO2 particles into a stainless steel capillary of inner diameter of 1.0 mm and length of 1.0 m. The TiO2 micro-packed column was connected to a conventional gas chromatograph equipped with a flame ionization detector or a thermal conductivity detector. The column showed high retentivity for carbon dioxide and also for organic compounds, achieving a baseline separation of methane and ethane. The TiO2 packed column was highly thermally stable, with a temperature limit above 400°C. Above 300°C, the analytes injected into the column were thermally degraded by catalytic combustion of TiO2 under N2 carrier gas. On the other hand, the degradation was obtained above 200°C using air as the carrier gas.
In order to explore the possibility of determining whether clenbuterol (CLB) was ingested unintentionally via meat products contaminated with CLB or taken intentionally for doping purposes by athletes, we conducted an in vitro study assuming in vivo chiral conversion of CLB. Enzymatic reaction using swine liver tissue or chemical reaction in artificial gastric juice was performed to clarify the chiral conversion of CLB enantiomers. LC/UV measurement revealed no chiral conversion in the enzymatic reaction and temperature-dependent chiral conversion in the artificial gastric juice where CLB finally racemized. From the calculated reaction rate, activation energy (Ea), and activation entropy (ΔS) in the chiral conversion reaction of R-CLB in artificial gastric juice, we expected that this chiral conversion would proceed slowly because Ea was relatively high. After heating at 38°C for 2 h, only approximately 1% of CLB underwent chiral conversion. Therefore, when humans ingest meat products contaminated with CLB having a different enantiomeric ratio, chiral conversion hardly progresses in the stomach and such ingestion would have very little effect on the enantiomeric excess of CLB excreted in urine. This suggests that measuring urinary CLB enantiomeric ratio would reveal whether CLB was ingested unintentionally via CLB-contaminated meat products or taken intentionally.
Upon absorption in the intestine of the host animal, the main function of short-chain fatty acids (SCFAs), mainly acetate, propionate and n-butyrate, is as metabolic energy. SCFAs, n-butyrate in particular, can also be found in the mouth. An excess of oral SCFAs may cause not only periodontal diseases but also systemic abnormalities in humans. Previously, we reported a method for simultaneous detection by gas chromatography-mass spectrometry (GC-MS) of acetate, propionate and n-butyrate in serum, urine and saliva. In the present study we used a modified version of this method to detect not only acetate, propionate and n-butyrate, but also iso-butyrate, n-valerate, iso-valerate and caproate, because the latter are suggested to be associated with periodontal diseases. Detection ranges of SCFAs were as follows; 6.25-400 µmol/L (acetate), 0.781-100 µmol/L (propionate and n-butyrate), 0.391-50 µmol/L (iso-butyrate), 0.781-50 µmol/L (n-valerate and iso-valerate) and 1.56-50 µmol/L (caproate). Furthermore, we validated the modified detection method with triple freeze-thaw-cycle recovery tests and intra- and inter-day repeatability. Freezing and thawing did not influence the concentrations of SCFAs in saliva. Upon analysis of five clinical saliva samples, it was observed that, except for n-valerate, which was detected only in two samples, all SCFAs were detected in saliva samples. To conclude, we were able to use a modified method to analyze successfully by GC-MS the salivary concentrations of SCFAs. In addition, we simultaneously detected the salivary concentrations of iso-butyrate, iso-valerate, nvalerate and caproate. This improved method was proved to be reliable to measure the concentrations of SCFAs in saliva.
An extraction capillary packed with a polyethylene terephthalate nanofiber-sheet was applied for the extraction of five water soluble polycyclic aromatic hydrocarbons (PAHs, including naphthalene (Nap), fluorene (Flu), phenanthrene (Phe), fluoranthene (Flt), and pyrene (Pyr)), in water samples. The extracted PAHs were eluted with acetonitrile, and on-line injected into a high-performance liquid chromatographic system coupled with a fluorescence detector. The limit of detections of the method for Nap, Flu, Phe, Flt, and Pyr were 1.0, 1.0, 0.2, 0.5, and 0.2 ng/mL, respectively, at a sample loading volume of 1.0 mL. The spiked recoveries of the analyte PAHs were in the range of 97.3 to 102% upon spiking the PAHs into tap water and river water samples.