Journal of Chromatography B (v.957, #C)
Editorial Board (i).
Simultaneous determination of Guanfu base G and its active metabolites by UPLC–MS/MS in rat plasma and its application to a pharmacokinetic study by Lingchun Zhang; Aiqin Wang; Xueqin Wang; Ying Zhang; Xiaotian Li; Ping Liu (1-6).
To establish a rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC–MS/MS) method for the determination of concentration of guanfu base G (GFG) and its active metabolites in rat plasma. The GFG and its active metabolites and the internal standard (phenacetin) were separated on an Acquity UPLC® BEH C18 chromatography column (2.1 mm × 50 mm I.D., 1.7 μm) using gradient elution with a mobile phase of methanol and ultrapure water at a flow rate of 0.4 mL/min. The detection was performed on a Xevo triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 472.26 → m/z 310.03 for GFG and m/z 180.00 → m/z 109.99 for phenacetin (IS) using a positive electrospray ionization interface. The lower limit of quantification (LLOQ) was 1 ng/mL, the limit of detection (LOD) was 0.3 ng/mL, and the linear calibration curve was obtained over the concentration range of 1–200 ng/mL. The intra-day and inter-day assay variations were measured to be below 10.97%, and the accuracy values (relative error) ranged from 95.4% to 103.6%. After validation, this method was successfully applied to a pharmacokinetic study where rats were intravenous administration of 5 mg/kg GFG. A simple, rapid, sensitive, and accurate method for the determination of the concentration of GFG and its metabolites in rat plasma was developed and validated.
Keywords: Guanfu base G; UPLC–MS/MS; Active metabolites; Rat plasma; Pharmacokinetic;
Packed-fiber solid-phase extraction coupled with high performance liquid chromatography–tandem mass spectrometry for determination of diethylstilbestrol, hexestrol, and dienestrol residues in milk products by Wen-Yan Hu; Xue-Jun Kang; Chi Zhang; Jun Yang; Rui Ling; E.-Hu Liu; Ping Li (7-13).
A sensitive analytical method based on packed-fiber solid-phase extraction and high performance liquid chromatography–tandem mass spectrometry (PF SPE-HPLC–MS/MS) has been developed for determination of three synthetic stilbenes in milk. The stilbenes are extracted with acetonitrile, using sodium chloride, and purified with PF SPE using a cartridge containing electrospun polystyrene nanofibers. Parameters affecting the efficiency of PF SPE, such as pH and amount of salt, were optimized. Under optimal conditions, the limits of detection and quantification were 5–13 pg/g and 15–37 pg/g, respectively. Absolute recoveries varied between 60% and 85% at three different levels. The method was successfully applied for the determination of estrogenic stilbenes in a total of 69 milk samples. The method is sensitive and cost-effective in stilbene detection, and has potential in quality control of dairy products.
Keywords: Liquid dairy products; Packed-fiber solid-phase extraction and high performance liquid chromatography–tandem mass spectrometry; Stilbenes;
Development and practical application of accelerated solvent extraction for the isolation of cocaine/crack biomarkers in meconium samples by Cínthia de Carvalho Mantovani; Marcela Bittar Lima; Carolina Dizioli Rodrigues de Oliveira; Rafael de Almeida Menck; Edna Maria de Albuquerque Diniz; Mauricio Yonamine (14-23).
A method using accelerated solvent extraction (ASE) for the isolation of cocaine/crack biomarkers in meconium samples, followed by solid phase extraction (SPE) and the simultaneous quantification by gas chromatography–mass spectrometry (GC–MS) was developed and validated. Initially, meconium samples were submitted to an ASE procedure, which was followed by SPE with Bond Elut Certify I cartridges. The analytes were derivatizated with PFP/PFPA and analyzed by GC–MS. The limits of detection (LOD) were between 11 and 17 ng/g for all analytes. The limits of quantification (LOQ) were 30 ng/g for anhydroecgonine methyl ester, and 20 ng/g for cocaine, benzoylecgonine, ecgonine methyl ester and cocaethylene. Linearity ranged from the LOQ to 1500 ng/g for all analytes, with a coefficients of determination greater than 0.991, except for m-hydroxybenzoylecgonine, which was only qualitatively detected. Precision and accuracy were evaluated at three concentration levels. For all analytes, inter-assay precision ranged from 3.2 to 18.1%, and intra-assay precision did not exceed 12.7%. The accuracy results were between 84.5 and 114.2% and the average recovery ranged from 17 to 84%. The method was applied to 342 meconium samples randomly collected in the University Hospital—University of São Paulo (HU—USP), Brazil. Cocaine biomarkers were detected in 19 samples, which represent 5.6% of exposure prevalence. Significantly lower birth weight, length and head circumference were found for the exposed newborns compared with the non-exposed group. This is the first report in which ASE was used as a sample preparation technique to extract cocaine biomarkers from a complex biological matrix such as meconium samples. The advantages of the developed method are the smaller demand for organic solvents and the minor sample handling, which allows a faster and accurate procedure, appropriate to confirm fetal exposure to cocaine/crack.
Keywords: Accelerated solvent extraction; Fetal exposure; Cocaine; Crack; Meconium; Gas chromatography–mass spectrometry;
Simultaneous quantification of the glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) receptor agonists in rodent plasma by on-line solid phase extraction and LC–MS/MS by Yan Wang; Jonathan D. Roth; Steven W. Taylor (24-29).
Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) and the cholecystokinin-1 receptor (CCK1-R) have therapeutic potential because of their marked anorexigenic and weight lowering effects. Furthermore, recent studies in rodents have shown that co-administration of these agents may prove more effective than treatment either of the peptide classes alone. To correlate the pharmacodynamic effects to the pharmacokinetics of these peptide drugs in vivo, a sensitive and robust bioanalytical method is essential. Furthermore, the simultaneous determination of both analytes in plasma samples by a single method offers obvious advantages. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is well suited to this goal through its ability to simultaneously monitor multiple analytes through selected reaction monitoring (SRM). However, it is a challenge to find appropriate conditions that allow two peptides with widely disparate physiochemical properties to be simultaneously analyzed while maintaining the necessary sensitivity for their accurate plasma concentrations. Herein, we report an on-line solid phase extraction (SPE) LC–MS/MS method for simultaneous quantification of the CCK1-R agonist AC170222 and the GLP-1R agonist AC3174 in rodent plasma. The assay has a linear range from 0.0975 to 100 ng/mL, with lower limits of quantification of 0.0975 ng/mL and 0.195 ng/mL for AC3174 and AC170222, respectively. The intra- and inter-day precisions were below 15%. The developed LC–MS/MS method was used to simultaneously quantify AC3174 and AC170222, the results showed that the terminal plasma concentrations of AC3174 or AC170222 were comparable between groups of animals that were administered with the peptides alone (247 ± 15 pg/mL of AC3174 and 1306 ± 48 pg/mL of AC170222), or in combination (222 ± 32 pg/mL and 1136 ± 47 pg/mL of AC3174 and AC170222, respectively). These data provide information on the drug exposure to aid in assessing the combination effects of AC3174 and AC170222 on rodent metabolism.
Keywords: Glucagon-like peptide-1 receptor; Cholecystokinin-1 receptor; LC–MS/MS; Solid phase extraction; Pharmacokinetics; Pharmacodynamics;
Quantitative analysis of glycerol levels in human urine by liquid chromatography–tandem mass spectrometry by Ying Dong; Yanhua Ma; Kuan Yan; Li Shen; Xiaobing Wang; Youxuan Xu; Genye He; Yun Wu; Jianghai Lu; Zhiyong Yang; Feifei Feng (30-35).
Glycerol has the latent capacity to act as a plasma volume expander and disguise blood doping practices. Therefore, it has been prohibited in sports as a masking agent by the World Anti-Doping Agency (WADA) since January 2010 and a urinary threshold (1 mg/mL) was recommended recently . The purpose of this study was to establish and validate a novel quantitative method for the determination of urinary glycerol concentrations using a liquid chromatography–tandem mass spectrometry approach. This simple yet highly specific method made use of the derivatization of glycerol by benzoyl chloride in aqueous solution at 40 °C followed by analysis via LC–ESI-MS/MS without sample pre-concentration or cleanup. The assay was linear over the concentration range of 1.0–1000 μg/mL for glycerol in human urine. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.3 μg/mL and 1.0 μg/mL, respectively. The intra- and inter-day precision of the method at three concentration levels (3, 500 and 900 μg/mL) was less than 12.2%. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity. The method has been successfully applied to the detection of glycerol in “Quality Assurance Program” samples provided by the World Association of Anti-Doping Scientists (WAADS) and routine doping-control samples in our laboratory.
Keywords: Glycerol; Quantitation; Derivatization; Doping-control; LC–ESI-MS/MS;
Determination of sitafloxacin in human plasma by liquid chromatography–tandem mass spectrometry method: Application to a pharmacokinetic study by Kai Huang; Jie Yang; Jing Zhang; Ying Ding; Lan Chen; Wen-Yan Xu; Xue-Jiao Xu; Ru Duan; Qing He (36-40).
A high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS/MS) method was developed and validated to determine sitafloxacin in human plasma with dextrorphan as internal standard. Chromatographic separation was performed on a ZORBAX SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with the mobile phase of methanol/water (containing 0.1% formic acid) (46:54, v/v) at a flow rate of 0.2 mL/min. Quantification was performed using multiple-reaction monitoring of the transitions at m/z 410.2 ⟶ 392.2 for sitafloxacin and m/z 258.1 ⟶ 157.1 for dextrorphan, respectively. The calibration curve was linear over the range of 5–2500 ng/mL with the lower limit of quantification of 5 ng/mL for sitafloxacin. The intra- and inter-day precisions were less than 8.3% and the deviations of assay accuracies were within ±4.1%. Sitafloxacin was sufficiently stable under all relevant analytical conditions. This method was successfully applied to the pharmacokinetic study of sitafloxacin in healthy Chinese volunteers.
Keywords: Sitafloxacin; Plasma concentration; LC–MS/MS; Pharmacokinetics;
Measurement of concentrations of whole blood levels of choline, betaine, and dimethylglycine and their relations to plasma levels by Hussain Mohamad Awwad; Susanne H. Kirsch; Juergen Geisel; Rima Obeid (41-45).
We aimed at developing a method for the measurement of choline and its metabolites in whole blood (WB). After an extraction step, quantification of choline, betaine, and dimethylglycine (DMG) was performed using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Plasma and WB metabolites were evaluated in a group of 61 elderly people. The calibration curves were linear (r 2 > 0.997) for all compounds. The inter- and intra-assay coefficients of variation for all analytes were <10%. The recoveries were >90% and the relative matrix effect were ≤4.0%. The median concentrations of choline, betaine, and DMG were 11.3, 27.8, and 5.9 μmol/L in plasma and 66.6, 165, and 13.7 μmol/L in WB, respectively. There were positive correlations between WB and plasma markers; for choline (r = 0.42), betaine (r = 0.61), and DMG (r = 0.56) (all p ≤ 0.001). The concentrations of betaine in WB and plasma were significantly higher in men than in women. The concentrations of WB choline and DMG did not differ significantly according to sex. In conclusion, we have established a reliable method for measuring choline metabolites in WB. The concentrations of WB choline, betaine, and DMG seem to reflect intracellular concentrations of these metabolites.
Keywords: Choline; Betaine; Dimethylglycine; Whole blood; Homocysteine;
Optimization of a chromatographic stationary phase based on gellan gum using central composite design by A.I.C. Gonçalves; L.A. Rocha; J.M.L. Dias; L.A. Passarinha; A. Sousa (46-52).
To develop a new stationary phase of easy production, low cost, biocompatible, biodegradable and low unspecific adsorption, a three-dimensional network was prepared by combining the natural polysaccharide of gellan with divalent cations. The stability of this cation exchange chromatographic matrix was optimized by using an experimental design tool. The optimal conditions proposed for the gellan gel formulation were 48 mM ZnSO4, 0% DMF, 25 °C, 0.75% gellan and 0.5 h. The applicability of gellan matrix was tested by chromatographic assays with three model proteins (bovine serum albumin (BSA), α-chymotripsin and lysozyme). The results showed that the retention occurred in function of the net charge of each protein in MES buffer pH 6.2 and the elution was performed by increase of ionic strength to 750 mM NaCl in MES buffer pH 6.2. Lysozyme was the more retained protein due to its positive charge more effective than α-chymotripsin, while BSA did not interact with the matrix due to its negative charge at these conditions. Dynamic binding capacity assays were accomplished to characterize this matrix and to compare with commercial resins. The values of dynamic binding capacity from gellan gel were 3.9 mg/mL and 17.4 mg/mL, at 10% and 50% of breakthrough, respectively. In this way, gellan gel might be a promising chromatographic matrix to explore ionic interactions and to be applied in different purification strategies, getting the best benefit from its use at low cost.
Keywords: Central composite design; Chromatography; Gellan gum; Ion exchange; Matrix;
Determination of sulfadiazine in Jiaozhou Bay using molecularly imprinted solid-phase extraction followed by high-performance liquid chromatography with a diode-array detector by Ziru Lian; Xingliang He; Jiangtao Wang (53-59).
A highly selective sample cleanup procedure featuring molecularly imprinted solid-phase extraction (MISPE) was developed for the isolation and determination of sulfadiazine (SDZ) in seawater samples from Jiaozhou Bay, China. The molecularly imprinted polymer (MIP) was prepared using SDZ as the template molecule and methacrylic acid as the functional monomer. The MIP was used as a selective sorbent for the solid-phase extraction of SDZ. An off-line MISPE method followed by high-performance liquid chromatography (HPLC) with diode-array detection was established for the analysis of SDZ. Good linearity of the MISPE column for SDZ standard solutions was obtained within 0–200 μg L−1 (R 2 > 0.99). The recoveries of spiked seawater samples were satisfactory as high as 88%. Finally, seven samples in Jiaozhou Bay were determined and there was no sulfadiazine found except #2 and #5 sample. The concentrations were respectively 0.33 μg L−1 and 0.28 μg L−1, and the relative standard deviations were 1.35% and 4.13% (n = 3).
Keywords: Sulfadiazine; Molecularly imprinted polymer; Solid-phase extraction; Seawater; Jiaozhou Bay;
Combination of aqueous two-phase extraction and cation-exchange chromatography: New strategies for separation and purification of alliin from garlic powder by Xiu-min Jiang; Yan-min Lu; Cong-ping Tan; Yan -Liang; Bo Cui (60-67).
In this study, a two-step process combining aqueous two-phase extraction (ATPE) with chromatography was developed for extraction and purification of alliin from garlic powder. The partition coefficient and yield value of alliin in different types of aqueous two-phase system (ATPS) were compared and response surface methodology (RSM) was used for analyzing and optimizing the extraction process. The optimal extraction conditions of 19% (w/w) (NH4)2SO4, 20% (w/w) 1-prpanol, at 30 °C, pH 2.35 with 8.54% (w/w) NaCl was chosen based on the higher yield. Compared to the results obtained with the conventional extraction method, this method had an evident advantage on yield (20.4 mg/g versus the original yield of 15.0 mg/g) and the concentration of alliin in extract solution by ATPE was close to three times of that with conventional extraction. The purification of alliin was carried out with the ammonium form of sulfonic acid cation-exchange resins 001 × 7. Sample solution with alliin concentration of 1 mg/mL was passed through resins and the desorption of alliin was accomplished by water at the flow velocity of 0.5 mL/min, 1.5 mL/min, respectively. The purity and recovery of alliin after purification were 80% and 76%, respectively.
Keywords: Alliin; Extraction; Purification; Aqueous two-phase extraction; Cation-exchange resin;
Characterization of in vitro metabolites of JWH-018, JWH-073 and their 4-methyl derivatives, markers of the abuse of these synthetic cannabinoids by Veniero Gambaro; Sebastiano Arnoldi; Stefania Bellucci; Eleonora Casagni; Lucia Dell’Acqua; Laura Fumagalli; Marco Pallavicini; Gabriella Roda; Chiara Rusconi; Ermanno Valoti (68-76).
In vitro incubation with human liver microsomes of JWH-018, JWH-073, JWH-122 and for the first time 1-butyl-3-(1-(4-methyl)naphthoyl)indole (the 4-methylnaphthoyl analogue of JWH-073) was investigated to identify the principal metabolites of alkylindole synthetic cannabinoids, thus helping the discovering of synthetic cannabinoids abusers. The results obtained showed that the most abundant metabolites were mono-hydroxylated derivatives either on the alkyl chain (ω or ω–1 position) or on the indole (presumably in position 5 or 6) and naphthalene moieties.Moreover the extraction conditions of these derivatives from biological fluids, mainly plasma and urine spiked with commercially available metabolite standards, and the incubation procedure were investigated to obtain a fast, reliable and suitable extraction protocol to detect either the parent drugs or their metabolites by means of GC/MS.
Keywords: Alkylindole metabolites; JWH-018; JWH-073; JWH-122 ;1-Butyl-3-(1-(4-methyl)naphthoyl)indole; GC/FID; GC/MS;
Quantitative analysis of 11-nor-9-carboxy-tetrahydrocannbinol (THC–COOH) in urine by LC–MS/MS following a simple filtration by Marc J. Rumpler (77-83).
Quantification methods utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS) are common in clinical and forensic toxicology laboratories and the efficiency and rapidity of such methods continues to evolve. In most cases, urine drug confirmation does not require a drug extraction and can quickly and easily be accomplished with a dilution followed by sample filtration. The report describes the validation of a simple confirmation method for 11-nor-9-carboxy-tetrahydrocannabinol (THC–COOH) and compared two types of filter extraction columns for sample clean-up. The method achieved a linear range of 10–3000 ng/mL, acceptable bias (−4.7–2.6%) and precision (0.9–6.9%) and autosampler stability up to 72 h. Universal filter columns offered less variable recovery over the linear range and fewer matrix interferences compared to THC–COOH specific filter columns. Authentic specimens testing positive for THC–COOH by LC–MS/MS were in good agreement with typically used GC–MS methods.
Keywords: THC–COOH; LC–MS/MS; Filtration;
Water-compatible magnetic imprinted microspheres for rapid separation and determination of triazine herbicides in environmental water by Fengxia Qiao; Kyung Ho Row; Mengge Wang (84-89).
A novel kind of water-compatible magnetic imprinted cyromazine microsphere (WCMM) was synthesized by water/oil/water suspension polymerization. The obtained WCMM was homogenously spherical with porous morphology and strong magnetic properties. The microspheres were successfully used as adsorbent in dispersive solid-phase extraction (WCMM-dSPE) to selectively extract cyanazine and atrazine from environmental water. Good linearity of the two analytes was observed in the range from 2.5 to 200.0 ng mL−1. The average recoveries at three spiking levels ranged from 84.8% to 104.3% with relative standard deviations (RSD) less than 6.9%. Compared with magnetic non-imprinted particles (WCMN), the proposed WCMM adsorbent of dSPE efficiently improved the efficiency of extracting cyanazine and atrazine from environmental water samples and eliminated the effect of cyromazine leakage on the quantitative analysis of cyanazine and atrazine. The proposed WCMM-dSPE method combined the advantages of magnetic separation, molecular imprinted microspheres and dSPE.
Keywords: Magnetic molecularly imprinted microspheres; Cyromazine; Dispersive solid-phase extraction; Cyanazine; Atrazine;
Application of high-speed counter-current chromatography combined with macroporous resin for rapid enrichment and separation of three anthraquinone glycosides and one stilbene glycoside from Rheum tanguticum by Tao Chen; Yong-Ling Liu; Chen Chen; Deng-Lang Zou; Jin-Mao You; Jing Sun; Yu-Lin Li (90-95).
In this paper, an efficient method was successfully established by the combination of macroporous resin (MR) and high-speed counter-current chromatography (HSCCC) for rapid enrichment and separation of aloe-emodin 8-O-β-D-glucoside, emodin 1-O-β-D-glucoside, emodin 8-O-β-D-glucoside and piceatannol 4′-O-β-D-(6″-O-gallate)-glucoside. Six kinds of macroporous resins were investigated in the first step and X-5 macroporous resin was selected for the enrichment of the target compounds. The recoveries of the target compounds reached 89.0, 85.9, 82.3 and 84.9% respectively after 40% ethanol elution. In the second step, the target compounds were separated by HSCCC with a two-phase solvent system composed of chloroform/ethyl acetate/methanol/water (8:1:6:5, v/v). The established method will be helpful for further characterization and utilization of Rheum tanguticum. The results demonstrate that MR coupled with HSCCC is a powerful technique for separation of bioactive compounds from natural products.
Keywords: Rheum tanguticum; Anthraquinone Glycoside; Stilbene glycoside; Macroporous resin; High-speed counter-current chromatography;
Simultaneous determination of four bioactive flavonoids from Polygonum orientale L. in dog plasma by UPLC–ESI-MS/MS and application of the technique to pharmacokinetic studies by Yong Huang; Peng Zhang; Feng He; Lin Zheng; Yong-lin Wang; Ji-zhou Wu (96-104).
The in vivo effects of traditional herbal medicines are generally mediated by multiple bioactive components. The main constituents of Polygonum orientale L. are flavonoids such as orientin, vitexin, cynaroside, and quercitrin. The aim of this study was to develop and validate a method for characterizing these flavonoids, in order to better understand the pharmacokinetics and pharmacodynamics of P. orientale L. We used ultra-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (UPLC–ESI-MS/MS) to analyze the flavonoids. After precipitation of the proteins with methanol, the flavonoids were separated on a BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 μm) by using an elution gradient of acetonitrile. Flavonoid content was determined using the multiple reaction monitoring (MRM) mode at m/z 449.2 → 329.2 for orientin, m/z 433.2 → 313.0 for vitexin, m/z 449.2 → 287.1 for cynaroside, m/z 449.2 → 303.4 for quercitrin, and m/z 417.0 → 267.0 for the internal standard, puerarin. Pharmacokinetics was assessed after intravenous administration of P. orientale L. extracts (POE) in Beagle dogs at a dose of 22, 44, or 88 mg/kg. Analysis of the standard curves by linear regression revealed high linearity over a 243-fold dynamic range for the four flavonoids (the lower limit of quantitation values were 4–21 ng/mL). The relative standard deviations of intra- and inter-day measurements were less than 15.1%, and the method was accurate to within −8.7% to 7.2%; the extraction recoveries from dog plasma were 70.6–89.3%, 69.8–88.7%, 72.5–85.7%, and 71.0–79.1% for orientin, vitexin, cynaroside, and quercitrin, respectively. Our results suggest non-linear pharmacokinetic characteristics with rapid clearance of the flavonoids. In conclusion, UPLC–ESI-MS/MS is a rapid and sensitive method for simultaneous quantification of multiple flavonoids from POE in dog plasma and is suitable for pharmacokinetic studies of herbal medicines.
Keywords: Polygonum orientale L. extracts; Flavonoids; UPLC–ESI-MS/MS; Beagle dog; Pharmacokinetics;
High performance liquid chromatographic method for the determination of cinepazide maleate and its application to a pharmacokinetic study in rats by Jinyi Zhao; Ying Song; Hujun Wang; Yuan Sun; Meiyou Liu; Chengtao Lu; Yan Li; Shan Wang; Xiaohe Zhu; Wenli Hai; Aidong Wen; Yanyan Jia (105-109).
A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated to quantify cinepazide maleate, a calcium blocker, in rat plasma. Cinepazide maleate and Tinidazole (internal standard) have been extracted by a simple liquid–liquid extraction before injection into chromatographic system. Chromatographic separation was achieved on a reversed phase C18 column with a mobile phase consisted of a water mixture of 10 mM potassium dihydrogen phosphate (pH = 4.5):methanol (40:60, v/v), pumped at flow rate of 1.0 mL/min, and detected at 303 nm. The method exhibited a linear range of 0.12–120 μg/mL in blank rat plasma, with the lower detection limit of 0.06 μg/mL. The method was statistically validated for linearity, accuracy, precision, selectivity and stability following FDA guidelines. The intra- and inter-assay coefficients of variation did not exceed ±15% from the nominal concentration. The accuracy of cinepazide maleate was within ±15% of the theoretical value. The assay has been applied successfully in a pharmacokinetic study of cinepazide maleate after a single intravenous at three doses in rat. And cinepazide maleate injection can improve the bioavailability of cinepazide maleate greatly, and has a dose-dependence profile in rats.
Keywords: Cinepazide maleate; HPLC; Pharmacokinetic; Method of validation;
Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by ultra high performance liquid chromatography tandem mass spectrometry and its application to a bioequivalence study by Xiangjun Qiu; Zhe Wang; Bing Wang; Hui Zhan; Xiaofeng Pan; Ren-ai Xu (110-115).
An ultra high performance liquid chromatography tandem mass spectrometry (U-HPLC–MS/MS) method was developed and validated to determine irbesartan (IRB) and hydrochlorothiazide (HCTZ) in human plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard losartan were separated on an Acquity U-HPLC BEH C18 column and mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the negative ion mode. The MRM transitions of m/z 427.2 → 206.9 and m/z 296.1 → 204.9 were used to quantify for IRB and HCTZ, respectively. The linearity of this method was found to be within the concentration range of 5–3000 ng/mL for IRB, and 0.5–300 ng/mL for HCTZ in human plasma, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for IRB and HCTZ in human plasma, respectively. The relative standard deviations (RSD) of intra and inter precision were less than 12% for both IRB and HCTZ. The analysis time of per sample was 2.5 min. The developed and validated method was successfully applied to a bioequivalence study of IRB (300 mg) with HCTZ (12.5 mg) tablet in Chinese healthy volunteers (N = 20).
Keywords: Irbesartan; Hydrochlorothiazide; U-HPLC–MS/MS; Human plasma; Bioequivalence;
Methodological considerations for the harmonization of non-cholesterol sterol bio-analysis by Dylan S. Mackay; Peter J.H. Jones; Semone B. Myrie; Jogchum Plat; Dieter Lütjohann (116-122).
Non-cholesterol sterols (NCS) are used as surrogate markers of cholesterol metabolism which can be measured from a single blood sample. Cholesterol precursors are used as markers of endogenous cholesterol synthesis and plant sterols are used as markers of cholesterol absorption. However, most aspects of NCS analysis show wide variability among researchers within the area of biomedical research. This variability in methodology is a significant contributor to variation between reported NCS values and hampers the confidence in comparing NCS values across different research groups, as well as the ability to conduct meta-analyses. This paper summarizes the considerations and conclusions of a workshop where academic and industrial experts met to discuss NCS measurement. Highlighted is why each step in the analysis of NCS merits critical consideration, with the hopes of moving toward more standardized and comparable NCS analysis methodologies. Alkaline hydrolysis and liquid–liquid extraction of NCS followed by parallel detection on GC-FID and GC–MS is proposed as an ideal methodology for the bio-analysis of NCS. Furthermore the importance of cross-comparison or round robin testing between various groups who measure NCS is critical to the standardization of NCS measurement.
Keywords: Non-cholesterol sterols; Phytosterols; GC-FID; GC–MS; Cholesterol metabolism;