Journal of Chromatography B (v.993-994, #C)

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme.
Keywords: Novel liquid/liquid extraction process; Surfactant; Acetonitrile; Recycling of phase components; Polygalacturonase; Durio zibethinus;

Enantioselective HPLC determination of oxiracetam enantiomers and application to a pharmacokinetic study in beagle dogs by Qiuyang Zhang; Wei Yang; Qing Zhang; Yue Yang; Junxiu Li; Yang Lu; Yi Zheng; Jiake He; Di Zhao; Xijing Chen (9-13).
An enantioselective high-performance liquid chromatography method was developed and validated for the determination of oxiracetam enantiomers, a cognition and memory enhancer, in beagle dog plasma. The plasma samples were prepared by methanol extraction from 200 μL plasma, and then the baseline resolution was achieved on a Chiralpak ID column (250 mm × 4.6 mm, 5 μm) with mobile phase of hexane–ethanol–trifluoroacetic acid (78:22:0.1, v/v/v) at flow rate of 1.0 mL/min. The column elute was monitored using ultraviolet detection at 214 nm. The method was linear over concentration range 0.50–100 μg/mL for both enantiomers. The relative standard deviation values for intra- and inter-day precision were 0.78–13.61 and 0.74–8.92% for (R)- and (S)-oxiracetam, respectively. The relative error values of accuracy ranged from −4.74 to 10.48% for (R)-oxiracetam and from −0.19 to 11.48% for (S)-oxiracetam. The method was successfully applied to a pharmacokinetic study of individual enantiomer and racemic oxiracetam in beagle dogs after oral administration. The disposition of the two enantiomers was not stereoselective and chiral inversion was not observed in beagle dogs. The pharmacokinetic profiles of (S)-oxiracetam were similar with racemic oxiracetam in beagle dogs.
Keywords: Oxiracetam; Stereoselective HPLC; Pharmacokinetics; Enantiomer; Racemate;

Detection of telomerase activity using microchip electrophoresis by Koji Karasawa; Hidetoshi Arakawa (14-19).
Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV = 0.67%) in the short time of 100 s.
Keywords: Microchip electrophoresis; Telomerase; Tumor marker; Hydroxypropyl methylcellulose; Polyethylene oxide; Diagnostic assay;

Analysis of nifedipine in human plasma and amniotic fluid by liquid chromatography-tandem mass spectrometry and its application to clinical pharmacokinetics in hypertensive pregnant women by Gabriela Campos de Oliveira Filgueira; Osmany Alberto Silva Filgueira; Daniela Miarelli Carvalho; Maria Paula Marques; Elaine Christine Dantas Moisés; Geraldo Duarte; Vera Lucia Lanchote; Ricardo Carvalho Cavalli (20-25).
Nifedipine is a dihydropyridine calcium channel blocker used for the treatment of hypertension in pregnant women. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for analysis of nifedipine in human plasma and amniotic fluid. Separation of nifedipine and nitrendipine (IS) was performed using a LiChroCART® RP-Select B column and a mixture of water:acetonitrile:glacial acetic acid (30:70:0.5 v/v) as the mobile phase. Aliquots of 500 μL of biological samples were extracted at pH 13 using dichloromethane:n-pentane (3:7 v/v). The validated method was applied to a study of the pharmacokinetics of nifedipine in human plasma and amniotic fluid samples collected up to 12 h after administration of the last slow-release nifedipine (20 mg/12 h) dose to 12 hypertensive pregnant women. The estimated pharmacokinetic parameters of nifedipine showed a mean AUC0-12 of 250.2 ng h/mL, ClT/F of 89.2 L/h, Vd/F of 600.0 L and t 1/2 5.1 h. The mean amniotic fluid/plasma concentration ratio was 0.05. The methods proved to be highly sensitive by showing a lower quantification limit of 0.1 ng/mL for both matrices. And this study reports for the first time the complete development and validation of the method to quantify nifedipine in amniotic fluid using LC-MS-MS.
Keywords: Nifedipine; LC-MS/MS; Pregnancy, Hypertensive; Pharmacokinetics;

This study reports on the development of a rapid, selective, and sensitive column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze sixteen drugs (antidepressants, anticonvulsants, anxiolytics, and antipsychotics) in plasma samples from schizophrenic patients. The developed organic-inorganic hybrid monolithic column with cyanopropyl groups was used for the first dimension of the column-switching arrangement. This arrangement enabled online pre-concentration of the drugs (monolithic column) and their subsequent analytical separation on an XSelect SCH C18 column. The drugs were detected on a triple quadrupole tandem mass spectrometer (multiple reactions monitoring mode) with an electrospray ionization source in the positive ion mode. The developed method afforded adequate linearity for the sixteen target drugs; the coefficients of determination (R 2) lay above 0.9932, the interassay precision had coefficients of variation lower than 6.5%, and the relative standard error values of the accuracy ranged from −14.0 to 11.8%. The lower limits of quantification in plasma samples ranged from 63 to 1250 pg mL−1. The developed method successfully analyzed the target drugs in plasma samples from schizophrenic patients for therapeutic drug monitoring (TDM).
Keywords: Column-switching; Organic–inorganic hybrid monolith; Liquid chromatography-tandem mass spectrometry; Plasma samples; Schizophrenia;

Pharmacokinetic of 5 components after oral administration of Fructus Forsythiae by HPLC-MS/MS and the effects of harvest time and administration times by Yang Bai; Jin Li; Wei Liu; Xiu-cheng Jiao; Jun He; Jiao Liu; Lin Ma; Xiu-mei Gao; Yan-xu Chang (36-46).
The unripe Fructus Forsythiae (Qingqiao) and ripe Fructus Forsythiae (Laoqiao) are two types of the clinical forms of commercial fructus of Forsythia suspensa(Thunb.) Vahl. There is limited information available for differences in pharmacokinetic properties of active components between unripe and ripe Fructus Forsythiae in vivo. A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of 9 typical components in rat plasma. The separation of nine analytes was performed on an Eclipse plus C18 (4.6 mm × 100 mm, 1.8 μm) column with the mobile phases consisted of a mixture of 0.1% formic acid in water and acetonitrile. Method validation indicated that the developed method was rapid, specific and sensitive. It was found that the AUC(0–24h) of 5 ingredients (forsythoside A, rutin, phillyrin, isorhamnetin and quercetin) in rats after single orally administrated unripe Fructus Forsythiae also had significant differences compared with those after single dose oral administration of ripe Fructus Forsythiae extract. The systemic exposure of 5 ingredients after multiple oral administration of Fructus Forsythiae extract had significantly increased than those after single oral administration. The results indicated that harvest time is not only effects the contents but the bioavailability of active components of Fructus Forsythiae, which suggests that the rate and extent of drug metabolism were altered when the clinical forms of commercial Fructus Forsythiae with different harvest time. The administration times could influence the bioavailability of active components of Fructus Forsythiae.
Keywords: Fructus Forsythiae; Forsythoside; Rutin; Phillyrin; LC-MS/MS;

A pre-clinical pharmacokinetic study in rats of three naturally occurring iridoid glycosides, Picroside-I, II and III, using a validated simultaneous HPLC–MS/MS assay by Jianwei Zhu; Bingyang Xue; Bo Ma; Qi Zhang; Ming Liu; Lei Liu; Di Yao; Huanhuan Qi; Yonglu Wang; Hanjie Ying; Zimei Wu (47-59).
A selective and sensitive high-performance liquid chromatography–electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the simultaneous quantitative determination of Picroside-I, II, and III in rat plasma and tissue homogenate to aid the pre-clinical studies. The chromatographic separation was performed on a Hypersil GOLD AQ C18 column using a gradient elution program with a mobile phase consisting of 2 mM ammonium acetate and acetonitrile. The detection was achieved using a triple quadrupole tandem MS in negative ionization multiple reaction monitoring (MRM) mode. One-step protein precipitation was selected for plasma and tissue sample preparation while liquid-liquid extraction failed to achieve satisfactory recoveries. The calibration curves of all three analytes in either plasma or tissue homogenate showed good linearity over the concentration range of 0.5–500 ng/mL with a limit of quantitation at 0.5 ng/mL. Both the intra- and inter-day accuracy and precision were within ±10%. The extraction recoveries were >70%, and the relative matrix effect ranged from 80.4% to 107.4% in all the biological samples. All the analytes were stable in matrices for at least 24 h at room temperature, or 21 days in frozen. Three freeze/thaw cycles did not cause degradation. The method was successfully applied for quantification of the three iridoid glycosides in the collected plasma and various tissues following intravenous administration in rats. Picroside-I, II, and III were all eliminated rapidly with large volume of distribution. Among the three glycosides, Picroside-II showed the highest liver uptake, and only Picroside-I and II were found to get across the blood brain barrier (BBB). These results were consistent with their hepatoprotective or neuroprotective effects reported clinically. With the aid of the efficient and reliable simultaneous LC-ESI-MS/MS assay this pharmacokinetic study provided insights into their therapeutic targets of these three iridoid glycosides as well as valuable experimental basis for an expansion of their clinical indications.
Keywords: LC-MS/MS; Picroside-I; Picroside-II; Picroside-III; Simultaneous determination; Pharmacokinetics; Tissue distribution;

An ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) method was developed to investigate the in vivo metabolism of 2-(2-hydroxypropanamido) benzoic acid (HPABA), a marine-derived anti-inflammatory drug, for the first time. Plasma, urine, feces and bile samples were collected from male and female rats after a single intragastric administration of HPABA at a dose of 100 mg/kg. Besides the parent drug, a total of 13 metabolites (3 phase I and 10 phase II metabolites) were detected and tentatively identified through comparing their mass spectrometry profiles with those of HPABA. Results demonstrated that metabolic pathways of HPABA in rats included decarboxylation, hydroxylation, dehydrogenation, glucuronidation, glycine conjugation and N-acetyl conjugation. In summary, this work provided valuable information regarding the metabolism of HPABA in rats, which would contribute to better understanding of its safety and mechanism of action.
Keywords: 2-(2-hydroxypropanamido) benzoic acid; UHPLC-FT-ICR-MS; Metabolism; Identification; Rat;

Liquid chromatography-tandem mass spectrometry for the quantification of flurbiprofen in human plasma and its application in a study of bioequivalence by Chenghan Mei; Bin Li; Qiangfeng Yin; Jing Jin; Ting Xiong; Wenjuan He; Xiujuan Gao; Rong Xu; Piqi Zhou; Heng Zheng; Hui Chen (69-74).
A simple, quick and accurate LC-MS/MS method for the quantification of flurbiprofen in human plasma with indomethacin as internal standard (IS) was developed and validated. Samples were treated with methanol to precipitate proteins, then separated on a Ultimate C18 column (5 μm, 2.1 × 50 mm) with a gradient elusion process. Mobile phase A was comprised of water and formic acid, mobile phase B was comprised of acetonitrile and formic acid. Multi reaction monitoring (MRM) signals were saved on a negative ionization electrospray mass spectrometer. The calibration curve showed good linearity in the range of 40.00–10000.00 μg/L (r 2  = 0.998). Intra-day RE was 0.2–2.2%. Inter-day RE was 0.5–3.4%. The samples showed good stability under the study conditions. No significant matrix effect was observed. The established method was then applied to a bioequivalence study of a flurbiprofen axetil formulation.
Keywords: Flurbiprofen; LC-MS/MS; Human plasma; Pharmacokinetics; Bioequivalence;

A rapid, sensitive and selective ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous determination of icariin, naringin and osthole in rat plasma. Plasma samples pretreatment involved a one-step liquid–liquid extraction with a mixture of ethyl acetate-methyl tert-butyl ether (3:1, ν/ν). The separation was performed on an ACQUITY UPLC™ BEH C18 column with a gradient mobile phase system of methanol and water. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM), with the transitions at m/z 513.3 → 366.8 (icariin), m/z 579.3 → 150.9 (naringin), m/z 245.1 → 189.0 (osthole) and m/z 237.1 → 194.1 (IS), respectively. A good linear response was observed over the concentration ranges of 1.06–424 ng/ml, 2.10–525 ng/ml and 1.05–1.05 × 103  ng/ml with lower limit of quantification (LLOQ) of 1.06, 2.10 and 1.05 ng/ml for icariin, naringin and osthole, respectively. The intra- and inter-day precisions (R.S.D.) were within 14.3%, and the accuracy (R.E.) ranged from −4.1% to 4.6% at three quality control levels. The sensitive and selective method was applied to a pharmacokinetic study of icarrin, naringin and osthole in rats after oral administration of Gushudan capsule.
Keywords: UPLC–MS/MS; Pharmacokinetics; Gushudan capsule; Icariin; Naringin; Osthole;

Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC–MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC–MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC–MS/MS (kappa value = 1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value = 1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC–MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC.
Keywords: Lecithin/sphingomyelin ratio; Fetal lung maturity; Cation-exchange resin; ESI-MS/MS; MALDI-TOF;

A simple and rapid ultra high performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed for the simultaneous determination of Atractylenolide I, II and III in rat plasma. Plasma samples were processed by liquid–liquid extraction with ethyl acetate, using schisandrin as internal standard (IS). Chromatographic separation was accomplished on a Thermo Hypersil GOLD C18 column (2.1 mm × 50 mm, 1.9 μm) with mobile phase consisting of acetonitrile and 0.1% formic acid–water (50:50, v/v). The detection was carried out by ESI-MS (positive ionization mode) and low-energy collision dissociation tandem mass spectrometric analyses using the multiple-reaction monitoring (MRM) scan mode. The quantification was performed using the transitions of the protonated molecule → product ion at m/z 231.0 → 185.1 for Atractylenolide I, at m/z 233.1 → 187.1 for Atractylenolide II and at m/z 249.1 → 231.1 for Atractylenolide III, respectively. Method validation revealed excellent linearity over investigated range together with satisfactory intra- and inter-day precision, accuracy, matrix effects and extraction recoveries. This method was successfully applied to the comparative pharmacokinetic study of Atractylenolide I, II and III in rat plasma after intragastric administration of Baizhufuling extract and Atractylodis extract.
Keywords: Atractylenolide; Atractylodis; Baizhufuling; Rat plasma; Pharmacokinetic; UPLC-MS/MS;