Journal of Chromatography B (v.878, #3-4)
Editorial Board (i).
HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids by Franciszek K. Główka; Katarzyna Kosicka; Marta Karaźniewicz-Łada (283-289).
11β-Hydroxysteroid dehydrogenase isoform 2 (11β-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11β-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard – prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C18 cartridges. The enzymatic hydrolysis of conjugated steroids was provided using β-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0–1000.0 ng mL−1, for allo-THF and THE + allo-THE 10.0–1000.0 ng mL−1. LOD (S/N = 3:1) for all analytes amounted 3.0 ng mL−1. Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0–12.1% and 9.2–14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0–174.5 ng mL−1 and 17.4–35.9 ng mL−1, respectively. Free urinary steroids were in the ranges: 12.0–54.1 μg/24 h (UFF) and 37.8–76.2 μg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11β-HSD2 activity in man.
Keywords: Urinary free cortisol; Urinary free cortisone; 11β-Hydroxysteroid dehydrogenase; 9-Anthroyl nitrile derivatization; Enzymatic hydrolysis;
Quantification of 4-aminopyridine in plasma by capillary electrophoresis with electrokinetic injection by Salvador Namur; Mario González-de la Parra; Gilberto Castañeda-Hernández (290-294).
A rapid and sensitive CE method for the determination of 4-aminopyridine in human plasma using 3,4-diaminopyridine as an internal standard was developed and validated. The analytes were extracted from 0.5-mL aliquots of human plasma by liquid–liquid extraction, using 8 mL of ethyl ether, and injected electrokinetically into capillary electrophoresis equipment. The instrumental conditions were obtained and optimized by Design of Experiments (DOE – factorial and response surface model), having as factors: separation voltage, ionic strength (buffer concentration), pH and temperature. The response variables were migration time, resolution, tailing factor and drug peak area. After obtaining mathematically predicted values for the response variables with best factors combinations, these were reproduced experimentally in good agreement with predicted values. In addition to optimal separation conditions obtained by Design of Experiments, sensitivity was improved using electrokinetic injection at 10 kV for 10 s, and a capillary with 50 cm effective length and 100 μm I.D. The final instrumental conditions were voltage at 19 kV, capillary temperature at 15 °C, wavelength at 254 nm, and phosphate buffer 100 mM, pH 2.5 as the background electrolyte. This assay was linear over a concentration range of 2.5–80 ng/mL with a lower limit of quantification of 2.5 ng/mL. The relative standard deviation for the assay precision was <7% and the accuracy was >95%. This method was successfully applied to the quantification of 4-aminopyridine (4-AP) in plasma samples from patients with spinal cord injury.
Keywords: 4-Aminopyridine; 3,4-Diaminopyridine; Plasma; Capillary electrophoresis; Pharmacokinetics; Pharmacodynamics; Electrokinetic injection;
Peptides surviving the simulated gastrointestinal digestion of milk proteins: Biological and toxicological implications by Gianluca Picariello; Pasquale Ferranti; Olga Fierro; Gianfranco Mamone; Simonetta Caira; Aldo Di Luccia; Stefano Monica; Francesco Addeo (295-308).
Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of β-lactoglobulin (β-Lg), and especially that of the peptide β-Lg f125–135, could have implications for the immunogenic action of β-Lg in the insurgence of cow's milk allergy.
Keywords: Milk proteins; Gastrointestinal digestion; Bioactive peptides; Cow's milk allergy; Mass spectrometry;
Application of displacement chromatography for the analysis of a lipid raft proteome by Maria Trusch; Alexandra Böhlick; Diana Hildebrand; Björn Lichtner; Andreas Bertsch; Oliver Kohlbacher; Sebastian Bachmann; Hartmut Schlüter (309-314).
Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC–MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC–MS approach.
Keywords: Displacement chromatography; 2-Dimensional LC/MS; Proteomics; Shotgun approach; Membrane proteins; Lipid rafts;
Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques by Niranjan M. Deshpande; Manish G. Gangrade; Maharudra B. Kekare; Vikas V. Vaidya (315-326).
Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.
Keywords: Amphotericin B; Natamycin; HPLC–MS–MS; Human plasma; Liposomal Amphotericin B; Free Amphotericin B; Solid phase extraction;
Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent by Yasuto Tsuruta; Kiyoshi Maruyama; Hirofumi Inoue; Keiko Kosha; Yuuko Date; Nobuyuki Okamura; Seiji Eto; Eijiro Kojima (327-332).
A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in borate buffer (20 mmol l−1, pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 °C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l−1, pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7–4.6% and 0.4–5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6–125 nmol (mg creatinine)−1 (mean ± SD: 21.6 ± 26.6 nmol (mg creatinine)−1, n = 20).
Keywords: Carnosine; Urine; Fluorescence detection; 4-(5,6-Dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride; HPLC;
Simultaneous determination of ABT-888, a poly (ADP-ribose) polymerase inhibitor, and its metabolite in human plasma by liquid chromatography/tandem mass spectrometry by Richard Wiegand; Jianmei Wu; Xianyi Sha; Patricia LoRusso; Jing Li (333-339).
A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid–liquid extraction by the addition of 0.25 ml of plasma with 10 μl of 1 M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C18 column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1–2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration–time profile of ABT-888 after its oral administration in cancer patients.
Keywords: ABT-888; PARP inhibitor; High performance liquid chromatography; Mass spectrometry; LC–MS/MS; Pharmacokinetics;
HPLC determination of novel dithiolethione containing drugs and its application for in vivo studies in rats by Daniela Giustarini; Elena Perrino; Valerio Tazzari; Ranieri Rossi (340-346).
A panel of new drugs obtained by grafting a sulfurated moiety, i.e. 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) onto existing drugs have been synthesized and their in vivo action is under preclinical evaluation. In the present paper we describe rapid HPLC methods to detect ADTOH derivatives of valproic acid (ACS2), sildenafil (ACS6), aspirin (ACS14) and diclofenac (ACS15) in plasma. These methods allow the simultaneous detection of the potential drugs and of ADTOH moiety. In the case of ACS14 the de-acetylated metabolite (ACS21) can also be concomitantly measured. The chromatographic separation was performed on a C18 column, applying a mobile phase consisting of a mixture of trifluoroacetic acid and acetonitrile. ADTOH, ACS6, ACS14, ACS21 were separated isocratically whereas ACS2 and ACS15 were separated applying gradient elution. The methods are precise and accurate, with a low quantification limit of 200 nM for ACS2, ACS15 and ACS21 or 100 nM for ADTOH, ACS6 and ACS14. The mean absolute recovery for all tested molecules was always found to be close to 100%. The methods are shown to be selective and linear in the range 0.2–50 μM and thus appear suitable for pharmacokinetic studies with ADTOH containing compounds, as indicated by exemplificative experiments performed with intravenous administration of the drugs to rats.
Keywords: Dithiolethione; Diclofenac; Valproic acid; Sildenafil; Aspirin;
High-performance ion chromatography method for separation and quantification of inositol phosphates in diets and digesta by K. Blaabjerg; J. Hansen-Møller; H.D. Poulsen (347-354).
A gradient high-performance ion chromatographic method for separation and quantification of inositol phosphates (InsP2–InsP6) in feedstuffs, diets, gastric and ileal digesta from pigs was developed and validated. The InsP2–InsP6 were separated on a Dionex CarboPac™ PA1 column using a gradient with 1.5 mol L−1 methanesulfonic acid and water. The exchange of the commonly used HCl with methanesulfonic acid has two advantages: (i) the obtained baseline during the separation is almost horizontal and (ii) it is not necessary to use an inert HPIC equipment as the methanesulfonic acid is not as aggressive as HCl. Twenty-three of the 27 separated inositol phosphate isomers were isolated. ICP-MS was used for quantification of phosphorus in the isolated isomers and used for calculation of correction factors for each isomer allowing InsP6 to be used as calibration standard. The detection limits for InsP2–InsP6 were in the range of 0.9–4.4 mg phosphorus L−1. The recovery of the major part of the inositol phosphates was 80–100%, and the CV for repeatability and reproducibility were 1–17% and 1–14%, respectively.
Keywords: Inositol phosphates; Phytate; Phytic acid; HPIC; Methanesulfonic acid; Correction factor;
Ultra-performance liquid chromatography–tandem mass spectrometry analysis of the bioactive components and their metabolites of Shaofu Zhuyu decoction active extract in rat plasma by Shulan Su; Jianming Guo; Jin-ao Duan; Tuanjie Wang; Dawei Qian; Erxin Shang; Yuping Tang (355-362).
A rapid, sensitive and selective ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometric (UPLC–ESI-MS/MS) method was developed for analysis and identification of the bioactive components and their metabolites in rat plasma following oral administration Shaofu Zhuyu decoction active extract. The analysis was carried out on an AcQuity™ UPLC chromatographic instrument and a QTOF mass spectrometer using positive and negative electrospray ionization (ESI), respectively. The results showed that sixteen peaks were detected and twelve peaks, including flavones, organic acids and terpene glycosides, were identified by comparing with reference compounds. Furthermore, nine metabolites, including quercetin glucuronide sulfates, quercetin diglucuronides, isorhamnetin sulfates, isorhamnetin glucosides, and isorhamnetin glucuronides were detected and identified in rat plasma based on the mass fragmentation behaviors and literature reports. These results provided a meaningful basis for evaluating the bioactive components and their action mechanisms of complex traditional Chinese medicines (TCMs).
Keywords: Shaofu Zhuyu decoction; Bioactive components; Metabolites; UPLC–QTOF-MS; Rat plasma;
Metabolites of puerarin identified by liquid chromatography tandem mass spectrometry: Similar metabolic profiles in liver and intestine of rats by Cheng-Feng Luo; Mu Yuan; Min-Sheng Chen; Shi-Ming Liu; Hong Ji (363-370).
Puerarin is a major active ingredient of Pueraria Radix. Puerarin may exert its medicinal functions in part via its metabolites. In this study, we identified these metabolites to better understand and elucidate puerarin's metabolic pathway. Puerarin was intravenously administered to rats and then metabolites in plasma samples were identified by rapid resolution liquid chromatography electrospray ionization-collision induced dissociation tandem mass spectrometry (RRLC-ESI-CID–MS/MS). Chromatography was conducted on a Zorbax SB C18 column (2.1 × 100 mm, 1.8 μm) at 30 °C, with a gradient mobile phase consisting of 0.05% formic acid and acetonitrile, a flow rate of 0.2 mL min−1, and a total run time of 14 min. MS/MS acquisition parameters were as follows: positive ionization mode, dry gas: nitrogen, 10 L min−1, dry temperature: 350 °C, nebulizer: 40 psi, capillary: −3500 V, scan range: 250–800. The autoMS, manual, or multiple reaction monitoring mode was selected as required. Two glucuronidated metabolites of puerarin (M1 and M2) were detected. M1 and M2 are presumed to be puerarin-7-O-glucuronide and puerarin-4′-O-glucuronide, respectively, and M2 likely is suspected to be the major metabolite because it represented the predominate peak. Kinetic studies of metabolites demonstrated that M1 and M2 were detected in rat plasma at 5 min after intravenous administration of puerarin, the levels of M1 and M2 then reached their peaks at 10–15 and 15–30 min, respectively. The metabolic profiles were similar in rat liver and intestine investigated by in situ liver and intestine perfusion, indicating that no metabolic regioselectivity of puerarin occurs in the two organs.
Keywords: Puerarin; Metabolites; Liquid chromatography tandem mass spectrometry; Glucuronidation;
Determination of d-saccharic acid-1,4-lactone from brewed kombucha broth by high-performance capillary electrophoresis by Kan Wang; Xuhua Gan; Xinyun Tang; Shuo Wang; Huarong Tan (371-374).
Kombucha is a health tonic. d-Saccharic acid-1,4-lactone (DSL), a component of kombucha, inhibits the activity of glucuronidase, an enzyme indirectly related with cancers. To date, there is no efficient method to determine the content of DSL in kombucha samples. In this paper, we report a rapid and simple method for the separation and determination of DSL in kombucha samples, using the high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD). With optimized conditions, DSL can be separated in a 50 cm length capillary at a separation voltage of 20 kV in 40 mmol/L borax buffer (pH 6.5) containing 30 mmol/L SDS and 15% methanol (v/v). Quantitative evaluation of DSL was determined by ultraviolet absorption at λ = 190 nm. The relationship between the peak areas and the DSL concentrations, in a specified working range with linear response, was determined by first-order polynomial regression over the range 50–1500 μg/mL with a detection limit of 17.5 μg/mL. Our method demonstrated excellent reproducibility and accuracy with relative standard deviations (RSD) of less than 5% DSL content (n = 5). This is the first report to determine DSL by HPCE. We have successfully applied this method to determine DSL in kombucha samples in various fermented conditions.
Keywords: Kombucha; d-Saccharic acid-1,4-lactone; High-performance capillary electrophoresis;
Analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultra high pressure liquid chromatography–heat assisted electrospray ionization–tandem mass spectrometry by Gunnar Boysen; Leonard B. Collins; Shengkai Liao; April M. Luke; Brian F. Pachkowski; Joanne L. Watters; James A. Swenberg (375-380).
Increased amounts of reactive oxygen species (ROS), generally termed oxidative stress, are frequently hypothesized to be causally associated with many diseases. Analyses of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in DNA and urine are widely used biomarkers for oxidative stress. Over the years it became clear that analysis of 8-oxo-dG in DNA is challenging due to artifactual formation during sample work up. The present study demonstrates that 8-oxo-dG can be measured reliably and accurately when appropriate precautions are taken. First, the presence of an antioxidant, metal chelator, or free radical trapping agent during sample preparation improves reproducibility. Second, sample enrichment by HPLC fraction collection was used to optimize sensitivity. Third, heat assisted electrospray ionization (HESI) eliminated potential interferences and improved assay performance and sensitivity. Subsequently, the UPLC–HESI–MS/MS method was applied to show the biphasic dose response of 8-oxo-dG in H2O2-treated HeLa cells. Application of this method to human lymphocyte DNA (n = 156) gave a mean ± SD endogenous amount of 1.57 ± 0.88 adducts per 106 dG, a value that is in agreement with the suggested amount previously estimated by European Standard Committee on Oxidative DNA Damage (ESCODD) and others. These results suggest that the present method is well suited for application to molecular toxicology and epidemiology studies investigating the role of oxidative stress.
Keywords: 8-Oxo-7,8-dihydro-2′-deoxyguanosine; Oxidative stress; LC–MS;
Identification of cardiac glycosides in fractions from Periploca forrestii by high-performance liquid chromatography/diode-array detection/electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance by Yong Li; Xianfu Wu; Jianbei Li; Yinghong Wang; Shishan Yu; Haining Lv; Jing Qu; Zeper Abliz; Jing Liu; Yuanyan Liu; Dan Du (381-390).
Cardiac glycosides are a class of naturally occurring compounds that are characterized by some interesting biological activities and are widely distributed in the plant kingdom and can also be found in some animals. There is an interest in the chemical characterization of these molecules due to their toxicity and their use in medicines. In the study reported here, a combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography equipped with diode-array detector (HPLC-DAD/ESI-MS n ), and hyphenation to both liquid chromatography and nuclear magnetic resonance spectroscopy (HPLC/NMR) were utilized for the on-line analyses of cardiac glycosides from Periploca forrestii. The fragmentation patterns and 1H NMR spectra of nine isolated cardiac glycosides were investigated; their fragmentation rules and 1H NMR spectral characteristics were summarized and applied to the structural identification of similar constituents in fractions from P. forrestii. As a result, a total of nine trace cardiac glycosides were tentatively determined by analyses of accurate molecular masses, representative fragment ions and characteristic 1H NMR signals provided by HPLC/high-resolution mass spectrometry (HRMS), HPLC-DAD/ESI-MS n and HPLC/1H NMR experiments, respectively. Of these, eight (2–9) are new compounds and one (1) is reported from P. forrestii for the first time. Results of the present study can benefit the rapid identification and targeted isolation of new cardiac glycosides from crude plant extracts.
Keywords: Periploca forrestii; Cardiac glycosides; High-performance liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry; Liquid chromatography/nuclear magnetic resonance; Structural characterization;
Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry by Sheelendra Pratap Singh; Wahajuddin; Dinesh K. Yadav; Preeti Rawat; Rakesh Maurya; Girish Kumar Jain (391-397).
A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.
Keywords: Formononetin; Daidzein; Rat plasma; Validation; LC–MS/MS; Pharmacokinetics;
Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection by M.I. Rodríguez Cáceres; A. Guiberteau Cabanillas; T. Galeano Díaz; M.A. Martínez Cañas (398-402).
A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g−1 for danofloxacin, from 25 to 100 ng g−1 for sarafloxacin and from 50 to 315 ng g−1 for difloxacin, respectively. The method presents detection limits under 10 ng g−1 and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques.
Keywords: Difloxacin; Sarafloxacin; Danofloxacin; HPLC; Electrochemical detection; Chicken tissues;
Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 μm sorbent by Joomi Ahn; Jonathan Bones; Ying Qing Yu; Pauline M. Rudd; Martin Gilar (403-408).
Separation by hydrophilic interaction chromatography (HILIC) with fluorescence detection utilizing a sub-2 μm glycan column for the separation of 2-aminobenzamide (2-AB) labeled N-linked glycans is described. The HILIC column packed with a 1.7 μm amide sorbent improves the peak capacity compared to a 3.0 μm HILIC column by a similar degree as observed in reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results indicated that the optimal peak capacity was achieved at flow rate 0.2–0.5 mL/min. HILIC method transfer guidelines were shown to further enhance the resolution of glycans by changing initial gradient conditions, flow rate, column temperature, and different column lengths. Additionally, excellent resolution can be achieved in the separation of 2-AB labeled glycans released from fetuin, RNase B, and human IgG with a rapid analysis time.
Keywords: Glycans; Oligosaccharides; Ultra-performance liquid chromatography; Hydrophilic interaction chromatography; 2-Aminobenzamide labeled glycans;
Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography–tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies by Adam S. Ptolemy; Emma Tzioumis; Arjun Thomke; Sami Rifai; Mark Kellogg (409-416).
Targeted analyses of clinically relevant metabolites in human biofluids often require extensive sample preparation (e.g., desalting, protein removal and/or preconcentration) prior to quantitation. In this report, a single ultra-centrifugation based sample pretreatment combined with a designed liquid chromatography–tandem mass spectrometry (LC–MS/MS) protocol provides selective quantification of 3,7-dimethylxanthine (theobromine) and 1,3,7-trimethylxanthine (caffeine) in human saliva, plasma and urine samples. The optimized chromatography permitted elution of both analytes within 1.3 min of the applied gradient. Positive-mode electrospray ionization and a triple quadruple MS/MS instrument operated in multiple reaction mode were used for detection. 13C3 isotopically labeled caffeine was included as an internal standard to improve accuracy and precision. Implementing a 20-fold dilution of the isolated low MW biofluid fraction prior to injection effectively minimized the deleterious contributions of all three matrices to quantitation. The assay was linear over a 160-fold concentration range from 2.5 to 400 μmol L−1 for both theobromine (average R 2 0.9968) and caffeine (average R 2 0.9997) respectively. Analyte peak area variations for 2.5 μmol L−1 caffeine and theobromine in saliva, plasma and urine ranged from 5 and 10% (intra-day, N = 10) to 9 and 13% (inter-day, N = 25) respectively. The intra- and inter-day precision of theobromine and caffeine elution times were 3 and <1% for all biofluids and concentrations tested. Recoveries for caffeine and theobromine ranged from 114 to 118% and 99 to 105% at concentration levels of 10 and 300 μmol L−1. This validated protocol also permitted the relative saliva, plasma and urine distribution of both theobromine and caffeine to be quantified following a cocoa intervention.
Keywords: Theobromine; Caffeine; Saliva; Plasma; Urine; Cocoa; LC–MS/MS;
Analysis of [2H7]methionine, [2H4]methionine, methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine by Yoshihiko Shinohara; Hiroshi Hasegawa; Tomoyoshi Kaneko; Yuka Tamura; Takao Hashimoto; Kimiyoshi Ichida (417-422).
Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [2H7]methionine, [2H4]methionine, methionine, [2H4]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [13C]Methionine and [13C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.
Keywords: GC–MS; Stable isotope; Methionine; Homocysteine; Pharmacokinetics;
Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples by Romain Guilhaumou; Caroline Solas; Angelique Rome; Madeleine Giocanti; Nicolas Andre; Bruno Lacarelle (423-427).
Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm × 2.0 mm, 3.0 μm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4 → 765.4 (m/z) for vincristine and 811.4 → 751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.
Keywords: Vincristine; Cancer; LC/MS/MS; Pharmacokinetics;
Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study by Pankaj Partani; Sanjay Gurule; Arshad Khuroo; Tausif Monif; Sandeep Bhardwaj (428-434).
A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.
Keywords: Cefuroxime; Tandem mass spectrometry; Solid phase extraction; Cefoxitin;
Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma by Gui-Zhong Xin; Jian-Liang Zhou; Lian-Wen Qi; Chang-Yin Li; Peng Liu; Hui-Jun Li; Xiao-dong Wen; Ping Li (435-441).
A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC–LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 μm particle size (Agilent Zorbax StableBond-C18, 4.6 mm × 50 mm, 1.8 μm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic–lipophilic balanced copolymer (2.1 mm × 20 mm, 25 μm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC–MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R > 0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.
Keywords: Turbulent-flow chromatography; Pharmacokinetic study; Traditional Chinese medicines; Verticine; Verticinone; Isoverticine;
Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS) by Hisaki Kojima; Minoru Inagaki; Tsuyoshi Tomita; Teruko Watanabe; Satoko Uchida (442-448).
A new approach for the separation and inline characterization of lipopolysaccharide (LPS) related compounds has been developed. The separation was based on the difference in the number of charged phosphate and ethanolamine groups, as non-stoichiometric substituents, on the polysaccharide backbone, and was achieved with reverse phase ion-paring chromatography (RPIP-HPLC). Tributylamine was used as an ion-pair reagent. In the conditions used in this study, tributylammonium then binds to the LPS related compounds through the negatively charged phosphate groups. This changes the hydrophobicity of the analytes at different positions and allows for separation based on both the number and position of the substituents on the analyte. The RPIP-HPLC was found to be effective for the separation of the O,N-deacylated derivative (deON) and polysaccharide portion (PS) from the LPS of Escherichia coli C strain. Post-column fluorescence derivatization (FLD), using sodium periodate and taurine, was used to detect the separated LPS related species. On the other hand, the separated species were also detected by direct infusion into the ESI-Q-MS using a volatile ammonium acetate buffer rather than the more traditional potassium phosphate buffer. The signal to noise ratio (S/N ratio) was low for the total ion chromatogram, however, high S/N ratios as well as good resolution were attained by selected ion monitoring (SIM) using m/z numbers corresponding to species with different numbers of non-stoichiometric substituents. Five species for deON and ten species for PS were clearly identified on the SIM chromatogram on the RPIP-HPLC/ESI-Q-MS. Accordingly, the present method allows for the effective separation and inline identification of the species corresponding to the diverse non-stoichiometric substitutions in LPS related compounds.
Keywords: Ion pairing-HPLC; Strongly basic anion-exchange chromatography; Lipopolysaccharide; Escherichia coli C; Mass spectrometry; Post-column fluorescence derivatization;
Volatile profiles of human skin cell cultures in different degrees of senescence by Cristian A. Acevedo; Elizabeth Y. Sanchez; Juan G. Reyes; Manuel E. Young (449-455).
It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.
Keywords: SPME; VOC; Skin cell; Senescence;
Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection by Lauriane Goldwirt; Aurélie Barrail-Tran; Maria Da Cruz; Anne-Marie Taburet; Valérie Furlan (456-460).
A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from −15.0 to −0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.
Keywords: Raltegravir; High-performance liquid chromatography; Photodiode array detection;
Simultaneous HPLC–UV analysis of rufinamide, zonisamide, lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy by Manuela Contin; Susan Mohamed; Carmina Candela; Fiorenzo Albani; Roberto Riva; Agostino Baruzzi (461-465).
We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 μL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 μm Hydro-RP, 250 mm × 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2–40 μg/mL for RFN and 2–80 μg/mL for ZNS. The limit of quantitation was 2 μg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).
Keywords: Rufinamide; Zonisamide; Antiepileptic drugs; High performance liquid chromatography; Deproteinization; UV detection;
Simultaneous determination of didanosine and its amino acid prodrug, valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats by Zhongtian Yan; Jin Sun; Jinling Wang; Youjun Xu; Yannan Chang; Ping Meng; Meng Zhu; Qiang Fu; Yongbing Sun; Zhonggui He (466-470).
A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.
Keywords: Ultra-performance liquid chromatography–tandem mass spectrometry; Didanosine; Valdidanosine; Solid-phase extraction; Hydrophilic interaction; Pharmacokinetic study;
Multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: Application to Quillaja saponins by Johan Bankefors; Lars I. Nord; Lennart Kenne (471-476).
A method for separation and detection of major and minor components in complex mixtures has been developed, utilising two-dimensional high-performance liquid chromatography (2D-HPLC) combined with electrospray ionisation ion-trap multiple-stage mass spectrometry (ESI-ITMS n ). Chromatographic conditions were matched with mass spectrometric detection to maximise the number of components that could be separated. The described procedure has proven useful to discern several hundreds of saponin components when applied to Quillaja saponaria Molina bark extracts. The discrimination of each saponin component relies on the fact that three coordinates (x, y, z) for each component can be derived from the retention time of the two chromatographic steps (x, y) and the m/z-values from the multiple-stage mass spectrometry (z n , n = 1, 2, …). Thus an improved graphical representation was obtained by combining retention times from the two-stage separation with +MS1 (z 1) and the additional structural information from the second mass stage +MS2 (z 2, z 3) corresponding to the main fragment ions. By this approach three-dimensional plots can be made that reveal both the chromatographic and structural properties of a specific mixture which can be useful in fingerprinting of complex mixtures.
Keywords: Chromatographic profiling; Metabolic analysis; Multiple-stage mass spectrometry; Quillaja saponins; Two-dimensional chromatography;
Capillary electrophoresis of chitooligosaccharides in acidic solution: Simple determination using a quaternary-ammonium-modified column and indirect photometric detection with Crystal Violet by Toshiaki Hattori; Nobuhiro Anraku; Ryo Kato (477-480).
Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 μM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC.
Keywords: Chitooligosaccharide; Electrophoretic mobility; Indirect photometric detection; Positively charged capillary column; Crystal Violet;
Enantiomeric determination of tramadol and O-desmethyltramadol in human plasma by fast liquid chromatographic technique coupled with mass spectrometric detection by Lukáš Chytil; Olga Matoušková; Olga Černá; Pavla Pokorná; Václav Vobruba; František Perlík; Ondřej Slanař (481-486).
A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for enantiomeric determination of tramadol and its primary phase metabolite O-desmethyltramadol in human plasma has been developed. Tramadol hydrochloride –13C, d3, was used as an isotopic labeled internal standard for quantification. The method involves a simple solid phase extraction. The analytes and internal standard were separated on Lux Cellulose-2 packed with cellulose tris(3-chloro-4-methylphenylcarbamate) using isocratic elution with hexane/isopropanol/diethylamine (90:10:0.1, v/v/v) at a flow rate of 1.3 mL/min. The APCI positive ionization mass spectrometry was used with multiple reaction monitoring of the transitions at m/z 264.2 → 58.2 for tramadol, m/z 250.1 → 58.2 for O-desmethyltramadol and m/z 268.2 → 58.2 for internal standard. Linearity was achieved between 1–800 ng/mL and 1–400 ng/mL (R 2 ≥ 0.999) for each enantiomer of tramadol and O-desmethyltramadol, respectively. Intra-day accuracies ranged among 98.2–102.8%, 97.1–109.1% and 97.4–102.9% at the lower, intermediate, and high concentration for all analytes, respectively. Inter-day accuracies ranged among 95.5–104.1%, 99.2–104.7%, and 94.2–105.6% at the lower, intermediate, and high concentration for all analytes, respectively. This assay was successfully used to determine the concentration of enantiomers of tramadol and O-desmethyltramadol in a pharmacogenetic study.
Keywords: Tramadol; O-Desmethyltramadol; Enantiomeric separation; Pharmacokinetics; Cytochrome P450 2D6;
Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS by Jens Martens-Lobenhoffer; Christin Dautz; Stefanie M. Bode-Böger (487-491).
Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.
Keywords: Cyclic guanosine monophosphate; Weak anion exchanger; Porous graphitic carbon; LC–MS/MS;
Performance of tiloronoxim and tilorone determination in human blood by HPLC–MS/MS: Method validation, uncertainty assessment and its application to a pharmacokinetic study by Xianhua Zhang; Jingli Duan; Suodi Zhai; Yiheng Yang; Li Yang (492-496).
A highly sensitive and selective HPLC–MS/MS method is presented for the quantitative determination of tiloronoxim and its metabolite tilorone in human blood. An aliquot of 200 μl human blood was extracted with a mixture of chloroform/ethyl ether (1/2, v/v), using metoprolol as the internal standard (the IS). Separation was achieved on an Xterra MS C18 column (50 mm × 2.1 mm, 5 μm) with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). Detection was performed using positive MRM mode on a TurboIonSpray source. The mass transitions monitored were m/z 426.3 → 100.0, m/z 411.3 → 100.0 and m/z 268.3 → 116.1 for tiloronoxim, tilorone and the IS, respectively. The method was fully validated using total error theory, which is based on β-expectation tolerance intervals and include trueness and intermediate precision. The method was found to be accurate over a concentration range of 1–100 ng/ml for both compounds. The measurement uncertainty based on β-expectation tolerance intervals was assessed at each concentration level of the validation standards. This method was successively applied to a pharmacokinetic study of tiloronoxim in healthy volunteers.
Keywords: Tiloronoxim; Tilorone; HPLC–MS/MS; Blood; Total error; Measurement uncertainty; Pharmacokinetics;
Validation and application of a liquid chromatography–tandem mass spectrometric method for quantification of the drug transport probe fexofenadine in human plasma using 96-well filter plates by Melonie L. Stanton; Melanie S. Joy; Reginald F. Frye (497-501).
A rapid method to determine fexofenadine concentrations in human plasma using protein precipitation in 96-well plates and liquid chromatography–tandem mass spectrometry was validated. Plasma proteins were precipitated with acetonitrile containing the internal standard fexofenadine-d6, mixed briefly, and then filtered into a collection plate. The resulting filtrate was diluted and injected onto a Phenomenex Gemini C18 (50 mm × 2.0 mm, 5 μm) analytical column. The mobile phase consisted of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (35:65, v/v). The flow rate was 0.2 ml/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization and high resolution multiple reaction monitoring mode (H-SRM). The linear standard curve ranged from 1 to 500 ng/ml and the precision and accuracy (intra- and inter-run) were within 4.3% and 8.0%, respectively. The method has been applied successfully to determine fexofenadine concentrations in human plasma samples obtained from subjects administered a single oral dose of fexofenadine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving fexofenadine.
Keywords: Fexofenadine; Fexofenadine-d6; Protein precipitation; LC–MS; Human plasma;
Development and validation of high-throughput liquid chromatography–tandem mass spectrometric method for simultaneous quantification of Clopidogrel and its metabolite in human plasma by Raghunadha Reddy S; Koteswara Rao.Divi; I. Sarath chandiran; K.N. Jayaveera; Y.K. Naidu; M.P. Kalyan Reddy (502-508).
A simple, sensitive and reliable method is described for simultaneous quantification of Clopidogrel and its metabolite in human plasma by using HTLC–MS/MS. The analytical procedure involves on-line coupling of extraction with Cyclone P (50 mm × 0.5 mm 50 μm) HTLC column by injecting 15 μL sample and chromatographic separation is performed with Cohesive Propel C18 (5 μm, 3.0 mm × 50 mm), followed by quantification with mass detector in SRM mode using ESI as an interface. The calibration curves were linear over a concentration range of 0.1–8 ng/mL of Clopidogrel and 70 ng/mL to 6 μg/mL of its metabolite using 20 mL human plasma per batch. The total run time of analysis was 7.5 min and the lower limits of quantification were 0.1 ng/mL for Clopidogrel and 70 ng/mL for its metabolite. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in bioavailability and bioequivalence study.
Keywords: Clopidogrel; Metabolite; HTLC; MS/MS; Human plasma; Validation;
Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the sensitive determination of folates in rice by Veerle De Brouwer; Sergei Storozhenko; Christophe P. Stove; Jeroen Van Daele; Dominique Van Der Straeten; Willy E. Lambert (509-513).
High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC–MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC–MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC–MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 μg/100 g and 0.12 and 0.91 μg/100 g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 μg/100 g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material.
Keywords: Folates; UPLC–MS/MS; Rice analysis; Biofortification;