Journal of Chromatography B (v.933, #C)

Determination of Pseudoginsengenin DQ in rat plasma by UPLC–MS/MS and application of the method in a pharmacokinetic study by Hongmei Gao; Zhuo Li; Pingya Li; Meiyu Lin; Liu Han; Fang Wang; Jinping Liu (1-7).
Pseudoginsengenin DQ (pseudoginsengenin of diol derivatives quest, PDQ), the product of the oxidative cyclization of protopanaxadiol, exhibits a significant pharmacological effect as an antiarrhythmic agent. A sensitive and rapid analytical method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) was initially developed for the detection of PDQ in rat plasma. Pre-treatment of the sample obtained from the plasma involved a single protein precipitation step, using methanol. PDQ and an internal standard (IS), physcion, were separated on a Waters ACQUITY UPLC BEH C18 analytical column (50 mm × 2.1 mm, 1.7 μm) using acetonitrile-0.1% formic acid in water (70:30, v/v) as the mobile phase, at a flow rate of 0.3 mL/min. Chromatography of the PDQ and IS was performed within 3 min. Detection was performed through positive ion electrospray ionization (ESI+) in multiple reaction-monitoring (MRM) mode. The assay was linear over the concentration range of 5–1000 ng/mL (r  > 0.9980). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.5 ng/mL and 5.0 ng/mL, respectively. The intra- and inter-day deviations (expressed as relative standard deviation, RSD) were ≤9.5% and ≤1.7%, respectively, and the accuracy (expressed as relative error, RE) was in the range of −1.1 to 2.7%. The recoveries of PDQ and IS were 95.2% and 100.7%, respectively, and the matrix effects were satisfactory in all of the biological matrices examined. This fully validated method was successfully applied to the pharmacokinetic study of rats after a single initial intragastric administration of 15 mg/kg PDQ. The main pharmacokinetic parameters: T max (the time to peak), C max (the concentration to peak), and t 1/2 (the biological half time) were 4.0 ± 0.0 h, 3265.12 ± 700.26 ng/mL, 5.97 ± 0.43 h, respectively.
Keywords: Pseudoginsengenin DQ; UPLC–MS/MS; Pharmacokinetics;

Ionic liquids (ILs) are a new type of reagent that has accelerated research in extraction technology. On the other hand, few studies have systematically applied 1-methylimidazole ([MIM]) series ILs to the extraction of bioactive compounds from plants. In this study, [MIM] series ILs were used to extract four bioactive flavonoids, such as dihydrokaempferol, quercitrin, amentoflavone and myricetin, from Chamaecyparis obtuse (CO) leaves. First, a screen of the extraction method and solvent revealed the [MIM] series ILs to be suitable as additives in methanol in Soxhlet extraction. Second, an examination of a range of cations and anions of [MIM] series ILs for extraction revealed 1-decyl-3-methylimidazolium bromide ([DMIM][Br]) to be the best selection as an additive in methanol for the Soxhlet extraction of flavonoids from (CO) leaves. Finally, some factors of extraction, such as temperature, time and amount of samples, were examined systematically using a response surface methodology (RSM). Based on the above optimization, 2.41, 3.47, 0.76 and 3.15 mg/g of dihydrokaempferol, quercitrin, amentoflavone and myricetin, respectively, were extracted from 15 g of CO leaves by 2.5 mg mL−1 of [DMIM][Br] as additives in 200 mL of methanol in Soxhlet extraction at 200 °C for 8 h. This study highlights the potential of [MIM] series ILs as promising reagents for the extraction of bioactive compounds from plants.
Keywords: 1-Methylimidazole series ionic liquids; 1-Decyl-3-methylimidazolium bromide; Bioactive compounds; Response surface methodology; Chamaecyparis obtuse;

Development of a simple method for simultaneous determination of nine subclasses of non-steroidal anti-inflammatory drugs in milk and dairy products by ultra-performance liquid chromatography with tandem mass spectrometry by Tao Peng; Ai-Ling Zhu; Yue-Ning Zhou; Ting Hu; Zhen-Feng Yue; Dong-Dong Chen; Guo-Min Wang; Jian Kang; Chun-lin Fan; Ying Chen; Hai-Yang Jiang (15-23).
A multi-residue analysis method for simultaneous determination of nine subclasses of non-steroidal anti-inflammatory drugs (NSAIDs) in milk and dairy products by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) has been established. The sample was initially extracted and deproteinized with ascorbic acid buffer (0.01 M, pH 3) and acetonitrile–ethyl acetate mixture, followed by centrifugation and evaporation, then reconstituted with acetonitrile-0.1% formic acid (1 + 1, v/v). After removal of lipid material by n-hexane, the sample was analyzed by UPLC–MS/MS with electro-spray ionization (ESI) interface in Multiple Reaction Monitoring (MRM) mode. The range of limits of detection (LODs) and limits of quantification (LOQs) were 0.03–0.30 μg/kg and 0.10–1.00 μg/kg, respectively. The recoveries in milk, milk powder, yogurt, processed cheese and milk beverage ranged from 61.7% to 117%, and the relative standard deviations (RSDs) were less than 17.9% at three spiked levels (1, 10 and 100 times of the LOQ). Matrix effects were also investigated and it was determined the signals of the analytes were suppressed from 9.4% to 76.6% in processed cheese. The proposed method was also applied to incurred sample analysis. The results proved that this method was suitable for the simultaneous determination of nine subclasses of NSAIDs residues in milk and dairy products.
Keywords: UPLC–MS/MS; Multi-residue analysis; NSAIDs; Milk; Dairy products;

A simple and sensitive HPLC method has been developed for the determination of aliskiren in human plasma through derivatization with 1-naphthyl isocyanate. The separation was achieved on a C18 column using a mobile phase consisting of acetonitrile/water/phosphoric acid (45:55:0.01, v/v/v, pH 3.2) in a flow rate of 1 mL/min with UV detection at 230 nm. Caffeine was used as an internal standard. The factors influencing the derivatization reaction yields were carefully studied and optimized. The method was linear over the concentration range of 5–400 ng/mL with a detection limit of 0.5 ng/mL and a limit of quantification of 1.0 ng/mL. The relative standard deviation was less than 4.2% for both intra-day assay and inter-day assay results. No interferences from endogenous compounds were encountered. The percentage recovery was in the range 97.1–98.6%. The method is suitable for routine therapeutic drug monitoring and for pharmacokinetic studies
Keywords: Aliskiren; HPLC; Naphthyl isocyanate derivatization; Plasma;

Modelling counter-current chromatography using a temperature dependence plate model by Yun Wei; Fengkang Wang; Shui Wang; Ying Zhang (30-36).
So far, research workers have built several math models of counter-current chromatography (CCC), such as cell model, eluting counter-current distribution model, continuous-stirred tank reactors model, and probability model. Based on plate theory and Van’t Hoff equation, a new temperature dependence plate model has been established in this paper. When temperature was taken into consideration, the error of retention time was significantly reduced. The relationship between temperature and partition coefficient K can be summarized into linear equations (log  K  =  A  −  B/T), which can be introduced to the plate model where A and B are constant and T is the temperature in Kelvin. K values decreased with the increasing temperature. Since resolution values (R) are related to K values, the change of temperature finally leads to the change of resolutions between different compounds. K values at different temperatures of a certain compound in a given system were calculated according to the equation above. New program with a temperature parameter was applied in prediction of experimental results, which diminished the error in modelling prediction. Besides, it is difficult for the CCC work when two compounds have similar K values. However, separation can be achieved by changing temperature, which can be directed by this new model.
Keywords: Counter-current chromatography; Partition coefficient; K values; Temperature dependence; Modelling;

Due to the lack of sensitivity in current methods for the determination of fenoterol (Fen), a rapid LC–MS/MS method was developed for the determination of (R,R′)-Fen and (R,R′;S,S′)-Fen in plasma and urine. The method was fully validated and was linear from 50 pg/ml to 2000 pg/ml for plasma and from 2.500 ng/ml to 160 ng/ml for urine with a lower limit of quantitation of 52.8 pg/ml in plasma. The coefficient of variation was <15% for the high QC standards and <10% for the low QC standards in plasma and was <15% for the high and low QC standards in urine. The relative concentrations of (R,R′)-Fen and (S,S′)-Fen were determined using a chirobiotic T chiral stationary phase. The method was used to determine the concentration of (R,R′)-Fen in plasma and urine samples obtained in an oral cross-over study of (R,R′)-Fen and (R,R′;S,S′)-Fen formulations. The results demonstrated a potential pre-systemic enantioselective interaction in which the (S,S′)-Fen reduces the sulfation of the active (R,R′)-Fen. The data suggest that a non-racemic mixture of the Fen enantiomers may provide better bioavailability of the active (R,R′)-Fen for use in the treatment of cardiovascular disease.
Keywords: Congestive heart failure; Bioavailability; Chirobiotic T; β2-Adrenergic receptor agonist;

LC-ESI-MS method for the determination of dexamethasone acetate in skin of nude mouse by Lingjun Li; Pengcheng Ma; Jun Wei; Kun Qian; Lei Tao (44-49).
A high-performance liquid chromatography-positive electrospray ionization single quadrupole mass spectrometric (LC-ESI-MS) method for the determination of dexamethasone acetate in skin of nude mouse using triamcinolone acetonide acetate as the internal standard (I.S.) was developed and fully validated. Both compounds were precipitated from skin homogenate with methanol and were separated by HPLC on a Shimadzu Shim-pack VP-ODS C18 column (150 mm × 2.0 mm, 5 μm) with a mobile phase of methanol–water (80:20, v/v) at a flow rate of 0.2 mL/min. Calibration curves were linear over the range of 0.05–5 μg/mL. The intra-run relative standard deviations were less than 9.59%. The inter-run relative standard deviations were less than 7.82%. The mean recovery was in the ranges of 89.95–95.97%, respectively. The method was successfully applied to determinate the concentration of dexamethasone acetate in skin and study the percutaneous absorption process in skin of nude mouse.
Keywords: Dexamethasone acetate; LC–MS; Determination; Skin; Nude mouse;

Tectorigenin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. It possesses hepatoprotective, estrogenic, hypoglycemic and anti-inflammatory activities. In the present study, the plasma pharmacokinetic profile of tectorigenin in rats was evaluated. We developed a selective and accurate U-HPLC/Q-TOFMS method for the simultaneous characterization of nine tectorigenin metabolites, and quantitation of six major metabolites in rat plasma, including tectorigenin-7-O-glucuronide-4′-O-sulfate (Te-7G-4′S), tectorigenin-di-O-sulfate (Te-diS), tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-4′-O-glucuronide (Te-4′G), tectorigenin-7-O-sulfate (Te-7S) and tectorigenin after oral administration of tectorigenin (130 mg/kg). The plasma concentrations reached maximal values of 6.20 ± 2.05 μmol/L at 0.96 ± 0.68 h for Te-7G-4′S, 4.42 ± 1.36 μmol/L at 1.92 ± 2.15 h for Te-diS, 33.50 ± 4.89 μmol/L at 0.75 ± 0.67 h for Te-7G, 3.28 ± 1.01 μmol/L at 0.75 ± 0.67 h for Te-4′G, 12.80 ± 2.80 μmol/L at 0.85 ± 1.54 h for Te-7S, and 12.0 ± 0.63 μmol/L at 0.23 ± 0.15 h for tectorigenin, respectively. Enterohepatic recirculation resulted in double or triple peaks concentration curve/time profiles of the metabolites. Since the total plasma concentrations of tectorigenin conjugated metabolites were much higher than that of the tectorigenin aglycone, an extensive phase II metabolism plays an important role in the pharmacokinetics of tectorigenin in vivo.
Keywords: Tectorigenin; Plasma pharmacokinetics; Glucuronidation; Sulfation; Rat; U-HPLC/Q-TOFMS;

In this study, ultra-performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF-MS) was applied to the rapid analysis of 20(S)-protopanaxadiol (PPD) metabolites in rats after oral administration, enabling the structural characterization of 23 metabolites in plasma, bile, urine, and feces. 16 of these, including M1–M5, M9, and M11–M15, have not been previously reported. The results also indicated that demethylation, dehydration, dehydrogenation, oxidation, deoxidation, and glucuronidation were the major metabolic reactions of PPD in vivo. This study provides important information about the metabolism of PPD which will be helpful for fully understanding its mechanism of action. Furthermore, structural modification of PPD in vivo may aid in obtaining new chemical derivatives for pharmacological screening.
Keywords: 20(S)-Protopanaxadiol; Metabolites; Rat; UPLC/TOF-MS;