Biomedical Chromatography

Li, Chia‐Ying; Chiu, Chiung‐Hui; Huang, Ho‐Shin; Lin, Chung‐Hua; Wu, Tian‐Shung

doi: 10.1002/bmc.572pmid: 16161181

A simple, sensitive and specific high‐performance liquid chromatographic method has been developed for the first time to simultaneously determine the eight major biologically active ingredients, namely paeoniflorin, naringin, sennoside A, baicalin, baicalein, saikosaponin a, rhein and emodin of the Chinese herbal formula Da‐Chai‐Hu‐Tang. The contents of these marker substances in Da‐Chai‐Hu‐Tang extract could be easily determined within 85 min. The assay was reproducible and accurate with overall intra‐day variations and accuracy of less than 2% and more than 97.9%, respectively. Results indicate that the developed HPLC assay can be successfully utilized as a quality control method for simultaneous determination of eight representative substances in Da‐Chai‐Hu‐Tang. Copyright © 2005 John Wiley & Sons, Ltd.

Zhang, Cuiying; Wang, Xuan; Shang, Mingying; Yu, Jie; Xu, Yuqiong; Li, Zhenguo; Lei, Liucheng; Li, Xiaomei; Cai, Shaoqing; Namba, Tsuneo

doi: 10.1002/bmc.565pmid: 16206137

An HPLC method was developed for the simultaneous determination of five aristolochic acids (AAs) and two aristololactams (ALs) in the following six Chinese drugs derived from Aristolochia species. Samples were analyzed on a C18 column with acetonitrile and 3.7 mm phosphoric acid buffer gradient elution, detected at 260 nm. Assay was linear over the range (µg/mL) 0.386–38.6 for aristolochic acid Va, 0.632–63.2 for aristolochic acid IVa, 0.200–20.0 for 9‐hydroxy aristolochic acid I, 0.352–35.2 for aristololactam II, 0.296–29.6 for aristolochic acid II, 0.274–27.4 for aristololactam I and 3.12–312 for aristolochic acid I. Average recoveries (%) of samples were 102.0, 95.9, 99.2, 102.2, 97.2, 97.1 and 97.8 for these seven constituents, respectively. The detection limit and retention time for the seven constituents ranged from 10.0 to 15.8 ng/mL and from 12 to 21 min. As a result of drug determination, contents (in mg/g) were as follows: AA‐I, 0.69–1.77; AA‐II, 0.02–0.18; 9‐OH AA‐I, 0.04–0.12; AA‐IVa, 0.76–3.36; AA‐Va, 0.04–0.31; AL‐I, 0.07–0.36; and AL‐II, 0.01–0.09 in Madouling; AA‐I, 0.03–0.41; AA‐II, 0.01–0.11; 9‐OH AA‐I, 0.00–0.60; AA‐IVa, 0.00–0.77; AA‐Va, 0.00–0.14; and AL‐I, 0.00–0.04 in Tianxianteng; AA‐I, 1.19–4.71; and AA‐II, 0.24–1.69 in Qingmuxiang; AA‐I, 2.79–5.48; AA‐II, 1.06–1.86; 9‐OH AA‐I, 0.01–0.09; AA‐IVa, 0.38–0.69; AA‐Va, 0.00–0.61; AL‐I, 0.00–0.02; and AL‐II, 0.00–0.02 in Bei‐madouling‐gen; AA‐I, 0.64–4.23; AA‐II, 0.06–0.40; and AA‐IVa, 0.08–0.25; in Guangfangji; and AA‐I, 1.88–9.72; AA‐II, 0.26–1.88; and AA‐IVa, 0.09–0.52 in Guanmutong. The other constituents were not detected in Tianxianteng, Qingmuxiang, Guangfangji and Guanmutong. Copyright © 2005 John Wiley & Sons, Ltd.

Zhong, Yan; Jiao, Zheng; Yu, Yunqiu

doi: 10.1002/bmc.566pmid: 16145659

A reliable and validated reversed‐phase high‐performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre‐column derivatization of valproic acid with 4‐bromomethyl‐6, 7‐dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50–30 µg/mL for MPA and 5.00–150 µg/mL for VPA. The relative standard deviations of the method of intra‐ and inter‐day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 µL). Copyright © 2005 John Wiley & Sons, Ltd.

Li, Lie; Sheng, Yu‐Xin; Zhang, Jin‐Lan; Wang, Shan‐Shan; Guo, De‐An

doi: 10.1002/bmc.567pmid: 16138297

A reversed‐phase liquid chromatographic method was used to determine the ginsenosides Rg1, Rb1 and Rd of Panax notoginseng in rat tissues (kidney, liver, heart, spleen and lung) after the administration of total saponins of P. notoginseng. The tissue samples were treated with solid‐phase extraction prior to HPLC. The calibration curves for the three saponins were linear in the given concentration ranges. The intra‐day and inter‐day assay coefficients in tissues were between 76 and 120% respectively. The recoveries of all the tissues were higher than 70%. This method was applied to evaluate the distribution of the three major saponins of P. notoginseng in rat tissues. Copyright © 2005 John Wiley & Sons, Ltd.

Schmidt, Andreas; Brune, Kay; Hinz, Burkhard

doi: 10.1002/bmc.568pmid: 16189813

Anandamide (N‐arachidonylethanolamine) is an endogenous cannabinoid receptor ligand that has been implicated in various physiological and pathophysiological functions. In the present study, a liquid–liquid extraction‐based reversed‐phase HPLC method with fluorometric detection was validated and applied for the analysis of anandamide in human plasma. Following derivatization with the fluorogenic reagent 4‐(N,N‐dimethylaminosulfonyl)‐7‐(N‐chloroformylmethyl‐N‐methyl‐amino)‐2,1,3‐benzoxadiazole (DBD‐COCl), the analyte was separated using an acetonitrile–water gradient at a flow rate of 0.8 mL/min, and spectrophotometric detection at 560 nm with an excitation wavelength of 450 nm. The retention times for anandamide and R(+)‐methanandamide (internal standard) were 27.1 and 30.7 min, respectively. The validated quantification range was 1–15 ng/mL. The developed procedure was applied to determine anandamide levels in human plasma following a 24 h incubation of human whole blood at 37°C in the presence or absence of phenylmethylsulfonyl fluoride, an inhibitor of the anandamide‐degrading enzyme fatty acid amide hydrolase. Anandamide levels determined under both conditions were within the validated concentration range with anandamide levels being 2.3‐fold higher in plasma from PMSF‐treated blood. Copyright © 2005 John Wiley & Sons, Ltd.

Redmann, Stefanie; Charles, Bruce G.

doi: 10.1002/bmc.569pmid: 16161186

The development and validation of a simple, rapid and selective high‐performance liquid chromatography (HPLC) method is described for the quantitation of itraconazole and hydroxy‐itraconazole in 100 µL of plasma from a paediatric population. The mobile phase of methanol (75% v/v) and water (25% v/v) was pumped at 1 mL/min through a C18 Symmetry™ (3.9 mm i.d. × 150 mm) cartridge. Using a protein‐precipitation method, 100 µL internal standard (IS) solution (R051012, 555 µg/L in acetonitrile) were added to 100 µL of plasma followed by 10 µL zinc sulphate solution (20% w/v). Itraconazole, hydroxy‐itraconazole and IS eluted at 4.7, 8.3 and 12.5 min, respectively and were detected ßuorometrically at 250 nm (excitation) and 380 nm (emission). Recoveries were 87.1–96.7%. Calibrations in drug‐free plasma were linear (r2 > 0.99) from 50 to 2000 µg/L, using 1/c2 (c = concentration) weighting. Intraday and interday imprecision (CV%) was 4.8–17.3 and 6.3–16.6% for itraconazole, and 4.6–17.9 and 7.02–18.4% for hydroxy‐itraconazole. Inaccuracy was −7.1 to −14.7% for itraconazole and −0.1 to −9.7% for hydroxy‐itraconazole. The clinical application of this method was demonstrated by measurement of itraconazole and hydroxy‐itraconazole in plasma samples drawn from paediatric cystic fibrosis patients, who were prescribed itraconazole for treatment of allergic bronchopulmonary aspergillosis. Copyright © 2005 John Wiley & Sons, Ltd.

Chawla, Sonia; Ghosh, Soma; Sihorkar, Vaibhav; Nellore, Ranjani; Kumar, T. R. Shantha; Srinivas, Nuggehally R.

doi: 10.1002/bmc.570pmid: 16161180

A simple, precise, accurate and rugged reversed‐phase high‐performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of five permeability model compounds, viz. antipyrine, metoprolol, ketoprofen, furosemide and phenol red. The method was intended to standardize rat in situ single‐pass intestinal perfusion studies to assess the intestinal permeability of drugs in the market as well as new chemical entities. Optimum resolution was achieved by gradient elution on a Symmetry Shield C‐18 analytical column with the mobile phase consisting of a mixture of aqueous potassium dihydrogen orthophosphate (pH 5.5; 0.01 m) and methanol at a flow rate of 1.5 mL/min. The retention times of antipyrine, metoprolol, ketoprofen, phenol red and furosemide were about 9, 12, 13, 16 and 17 min, respectively. Data acquisition was carried out using a photo diode array detector in the wavelength range 210–600 nm. Extraction of chromatograms was carried out by timed wavelength. Data obtained in all studies indicated that the method was suitable for the intended purpose. The validated method was found to be linear and precise in the working range. Suitability of storage under various conditions and freeze/thaw impact at cold temperature were established to ensure complete sample recovery without any stability issues. Recovery very close to the spiked amounts indicated that the method was highly accurate and suitable for use on routine basis. Copyright © 2005 John Wiley & Sons, Ltd.

Tsukamoto, Yuhki; Santa, Tomofumi; Yoshida, Hiroo; Miyano, Hiroshi; Fukushima, Takeshi; Hirayama, Kazuo; Imai, Kazuhiro; Funatsu, Takashi

doi: 10.1002/bmc.574pmid: 16167303

Al‐Dirbashi, Osama Y.; Aboul‐Enein, Hassan Y.; Al‐Odaib, Ahmed; Jacob, Minnie; Rashed, Mohamed S.

doi: 10.1002/bmc.571pmid: 16167302

A liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the determination of ziprasidone (ZIP) in human plasma was developed. ZIP and N‐methyl ziprasidone as internal standard (IS) were extracted from alkalinized plasma using tert‐ butyl methyl ether. Separation was performed isocratically on a C8 column with 90% acetonitrile containing 2 mmol/L ammonium acetate as a mobile phase with a total run time of 2.5 min. MS/MS transitions of m/z 413 → 194 and m/z 427 → 177 of the analyte and internal standard were used for quantification. Confirmatory ions of m/z 413 → 177 and m/z 427 → 180 were collected as well. The calibration curve based on peak‐area ratio was linear up to at least 200 ng/mL with a detection limit of 0.1 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 5%. The method was successfully applied to the analysis of ZIP in spiked human plasma. Copyright © 2005 John Wiley & Sons, Ltd.

Urban, V. M.; Cass, Q. B.; Oliveira, R. V.; Giampaolo, E. T.; Machado, A. L.

doi: 10.1002/bmc.575pmid: 16177959

Two high‐performance liquid chromatographic methods for determination of residual monomer in dental acrylic resins are described. Monomers were detected by their UV absorbance at 230 nm, on a Nucleosil® C18 (5 µm particle size, 100 Å pore size, 15 × 0.46 cm i.d.) column. The separation was performed using acetonitrile–water (55:45 v/v) containing 0.01% triethylamine (TEA) for methyl methacrylate and butyl methacrylate, and acetonitrile–water (60:40 v/v) containing 0.01% TEA for isobutyl methacrylate and 1,6‐hexanediol dimethacrylate as mobile phases, at a flow rate of 0.8 mL/min. Good linear relationships were obtained in the concentration range 5.0–80.0 µg/mL for methyl methacrylate, 10.0–160.0 µg/mL for butyl methacrylate, 50.0–500.0 µg/mL for isobutyl methacrylate and 2.5–180.0 µg/mL for 1,6‐hexanediol dimethacrylate. Adequate assay for intra‐ and inter‐day precision and accuracy was observed during the validation process. An extraction procedure to remove residual monomer from the acrylic resins was also established. Residual monomer was obtained from broken specimens of acrylic disks using methanol as extraction solvent for 2 h in an ice‐bath. The developed methods and the extraction procedure were applied to dental acrylic resins, tested with or without post‐polymerization treatments, and proved to be accurate and precise for the determination of residual monomer content of the materials evaluated. Copyright © 2005 John Wiley & Sons, Ltd.