Journal of Chromatography B (v.878, #1)
Editorial Board (i).
Structural characterization of trace stilbene glycosides in Lysidice brevicalyx Wei using liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry by Youcai Hu; Jing Qu; Yuanyan Liu; Shishan Yu; Jianbei Li; Jinlan Zhang; Dan Du (1-7).
The mass fragmentation patterns of stilbene glycosides isolated from the genus Lysidice were investigated by negative ion electrospray ionization tandem mass spectrometry, and the influence of collision energy on their fragmentation behavior is discussed. It is found that the presence of the Y0 − and B0 − ions in the MS2 spectra is characteristic for 1 → 6 linked diglycosyl stilbenes, while the Y0 −, Y1 −, and Z1 − ions are representative ions of 1 → 2 linked diglycosyl stilbenes. These results indicate that ESI-MS n in the negative ion mode can be used to differentiate 1 → 6 and 1 → 2 linked diglycosyl stilbenes. Based on the fragmentation rules, 9 new trace constituents were identified or tentatively characterized in a fraction of Lysidice brevicalyx by using HPLC/HRMS and HPLC-DAD/ESI-MS n . The results of the present study can assist in on-line structural identification of analogous constituents and targeted isolation of novel compounds from crude plant extracts.
Keywords: Lysidice brevicalyx; Stilbene glycoside; HPLC-DAD/ESI-MS n ; Fragmentation behavior; On-line identification;
HPLC analysis of asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH by Catherine E. Jones; Christabelle J. Darcy; Tonia Woodberry; Nicholas M. Anstey; Yvette R. McNeil (8-12).
There is increasing recognition of the clinical importance of endogenous nitric oxide synthase inhibitors in critical illness. This has highlighted the need for an accurate high performance liquid chromatography (HPLC) method for detection of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in small volumes of blood. Here, the validation of an accurate, precise HPLC method for the determination of ADMA, SDMA, homoarginine and arginine concentrations in plasma is described. Solid phase extraction is followed by derivatisation with AccQ-Fluor™ and reversed phase separation on a Gemini-NX column at pH 9. Simultaneous detection by both UV–vis and fluorescence detectors affords extra validation. This solid phase extraction method gives absolute recoveries of more than 85% for ADMA and SDMA and relative recoveries of 102% for ADMA and 101% for SDMA. The intra-assay relative standard deviations are 2.1% and 2.3% for ADMA and SDMA, respectively, with inter-assay relative standard deviations of 2.7% and 3.1%, respectively. Advantages of this method include improved recovery of all analytes using isopropanol in the solid phase extraction; sharp, well-resolved chromatographic peaks using a high pH mobile phase; a non-endogenous internal standard, n-propyl l-arginine; and accurate and precise determination of methylated arginine concentrations from only 100 μL of plasma.
Keywords: Asymmetric dimethylarginine; Symmetric dimethylarginine; Homoarginine; Arginine; High performance liquid chromatography;
Confirmatory analysis of buprenorphine, norbuprenorphine, and glucuronide metabolites in plasma by LCMSMS. Application to umbilical cord plasma from buprenorphine-maintained pregnant women by Marta Concheiro; Hendreé Jones; Rolley E. Johnson; Diaa M. Shakleya; Marilyn A. Huestis (13-20).
An LCMSMS method was developed and fully validated for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) in 0.5 mL plasma, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. Transitions monitored were 468.3 > 396.2 and 468.3 > 414.3 for BUP, 414.3 > 340.1 and 414.3 > 326.0 for NBUP, 644.3 > 468.1 and 644.3 > 396.3 for BUP-Gluc, and 590.3 > 414.3 and 590.3 > 396.2 for NBUP-Gluc. Linearity was 0.1–50 ng/mL for BUP and BUP-Gluc, and 0.5–50 ng/mL for NBUP and NBUP-Gluc. Intra-day, inter-day, and total assay imprecision (%RSD) were <16.8%, and analytical recoveries were 88.6–108.7%. Extraction efficiencies ranged from 71.1 to 87.1%, and process efficiencies 48.7 to 127.7%. All compounds showed ion enhancement, except BUP-Gluc that demonstrated ion suppression: variation between 10 different blank plasma specimens was <9.1%. In six umbilical cord plasma specimens from opioid-dependent pregnant women receiving 14–24 mg/day BUP, NBUP-Gluc was the predominant metabolite (29.8 ± 7.6 ng/mL), with BUP-Gluc (4.6 ± 4.8 ng/mL), NBUP (1.5 ± 0.8 ng/mL) and BUP (0.4 ± 0.2 ng/mL). Although BUP biomarkers can be quantified in umbilical cord plasma in low ng/mL concentrations, the significance of these data as predictors of neonatal outcomes is currently unknown.
Keywords: Buprenorphine; Plasma; LCMSMS; Umbilical cord plasma;
Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS3 on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study by Nicola Cesari; Stefano Fontana; Dino Montanari; Simone Braggio (21-28).
Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS3 scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS3 scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r 2 = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS3) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.
Keywords: Protein precipitation; d-Amphetamine; LC–MS/MS/MS; Pharmacokinetics;
A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats by Ying-yong Zhao; Xian-long Cheng; Yongmin Zhang; Xu Chao; Ye Zhao; Rui-chao Lin; Wen-ji Sun (29-33).
A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)+ 393–268 for ergone and (m/z)+ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.
Keywords: Ergosta-4,6,8(14),22-tetraen-3-one; HPLC–APCI-MS/MS; Pharmacokinetics;
Determination of cadmium, cobalt, copper, iron, manganese, and zinc in thyroid glands of patients with diagnosed nodular goitre using ion chromatography by Anna Błażewicz; Wojciech Dolliver; Suceil Sivsammye; Apeksha Deol; Rajinder Randhawa; Grażyna Orlicz-Szczęsna; Remigiusz Błażewicz (34-38).
The aim of this study was to estimate and compare the contents of selected metals in 65 pathological (diagnosed nodular goitre) and 50 healthy human thyroid tissues (taken during autopsies). Ion chromatography (IC) preceded by microwave mineralization was applied for the first time for determination of cadmium, cobalt, copper, iron, manganese, and zinc in human thyroid samples. The study proved that the concentrations of Cu2+, Mn2+, Fe3+, and Zn2+ were significantly higher in the control group (healthy thyroids) in comparison with the studied group (nodular goitre) (p < 0.05), whereas for Co2+ the difference between two means of concentration (healthy vs pathological thyroids) was not significant statistically at 0.05 significance level. Measurement accuracy was verified by measurements of NIST standard reference material (1566a Oyster Tissue). Very good precision (RSD below 5%) and recoveries (above 90%) were evaluated.
Keywords: Thyroid; Nodular goitre; Metal ions; Ion chromatography;
Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS by J.W.C. Alffenaar; A.M.A. Wessels; K. van Hateren; B. Greijdanus; J.G.W. Kosterink; D.R.A. Uges (39-44).
Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.
Keywords: LC/MS/MS; Therapeutic drug monitoring; Pharmacokinetics; Azole antifungal agent; Routine;
Simple assay of plasma sevoflurane and its metabolite hexafluoroisopropanol by headspace GC–MS by Daniel Bourdeaux; Valérie Sautou-Miranda; Agnès Montagner; Sébastien Perbet; Jean Michel Constantin; Jean-Etienne Bazin; Jean Chopineau (45-50).
The anesthetic sevoflurane can now be delivered over periods of up to 48 h using a newly developed medical system, the AnaConDa (anesthetic conserving device). Lack of pharmacokinetic data on sevoflurane and its main metabolite (hexafluoroisopropanol, HFIP) in this indication prompted us to develop a headspace GC–MS method to quantify the two substances. The only previously published method for assaying the two substances could not be adapted to our study since it uses expensive and rarely employed system components together with toxic carbon disulfide as a dilution solvent. The method developed is straightforward and uses the relatively non-toxic solvent undecane as dilution solvent and chloroform as internal standard. The method is linear for a concentration range of 1–150 μg/ml, and presents high accuracy and precision. LOD and LOQ are 0.2 and 1 μg/ml, with a short analysis time (7.6 min for a single analysis). The method was applied to determine the plasma levels of sevoflurane and HFIP in six patients under 48-h anesthetic sedation delivered via the AnaConDa system. Average sevoflurane and HFIP concentrations plateaued at 75 and 4 μg/ml, respectively. Sevoflurane quickly tailed off after inhalation was stopped, and HFIP levels remained low.
Keywords: Sevoflurane; HFIP; Headspace gas chromatography; Mass spectroscopy; Pharmacokinetic study; Anesthetic conserving device;
High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma by Xiaoqiang Xiang; Yi Han; Mikko Neuvonen; Jouko Laitila; Pertti J. Neuvonen; Mikko Niemi (51-60).
We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.
Keywords: Bile acids; Liquid chromatography–mass spectrometry; Healthy subjects; Bile acid-free plasma; Bile acid stability;
HPLC-based lipophilicity of pyrrolyl-acetic acid ARIs: Relationships with biological activity by Marios Chrysanthakopoulos; Ioannis Nicolaou; Vassilis J. Demopoulos; Anna Tsantili-Kakoulidou (61-67).
Reversed phase HPLC was used to assess the lipophilicity of a series pyrrolyl-acetic acid derivatives with aldose reductase inhibitory activity. The pH conditions were adjusted at 3.0 to investigate the behavior of the neutral species and at pH 7.4, at which the ionized form predominates, using phosphate and MOPS buffer. Retention was monitored in absence and in presence of different amounts of n-octanol in the mobile phase in order to explore the chromatographic conditions which best reproduce the octanol–water partition or distribution coefficients. The effect of n-octanol in retention was systematically studied and its role in lipophilicity assessment was evaluated. Nevertheless rather moderate regression equations were obtained, which deviated significantly from the ideal 1:1 correlation. No significant effect of buffer was observed. The appropriateness of retention factors to be used in correlation with aldose reductase inhibitory activity was further evaluated and compared to the efficiency of the corresponding octanol–water log P values.
Keywords: Pyrrolyl-acetic acid derivatives; Lipophilicity; RP-HPLC; n-Octanol as mobile phase additive; Aldose reductase inhibitory activity;
Quantification of key folate forms in serum using stable-isotope dilution ultra performance liquid chromatography–tandem mass spectrometry by Susanne H. Kirsch; Jean-Pierre Knapp; Wolfgang Herrmann; Rima Obeid (68-75).
Folates act as essential coenzymes in many biological pathways. Alteration in folate form distribution might have biological significance, especially in relation to certain genetic polymorphisms. We developed a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for quantification of the folate forms 5-methyltetrahydrofolate (5-methylTHF), 5-formylTHF, 5,10-methenylTHF, THF, and folic acid in serum. After extraction using an ion exchange and mixed mode solid-phase, samples were separated and detected using an UPLC–MS/MS system. The quantification limits were between 0.17 nmol/L (5-formylTHF) and 1.79 nmol/L (THF), and the assay was linear up to 100 nmol/L (5-methylTHF) and 10 nmol/L (5-formylTHF, 5,10-methenylTHF, THF, and folic acid). The intraassay CVs for 5-methylTHF and 5-formylTHF were 2.0% and 7.2%, respectively. Mean recoveries were between 82.3% for THF and 110.8% for 5,10-methenylTHF. Concentrations of total folate measured by the new method showed a strong correlation with those measured by an immunologic assay (r = 0.939; p < 0.001). The mean total folate from 32 apparently healthy subjects was 18.09 nmol/L, of which 87.23% was 5-methylTHF. Concentrations of homocysteine showed a better correlation to the total folate measured by the new method compared to that obtained by an immunologic assay. We also confirmed that MTHFR polymorphism has a significant effect on folate distribution in this small population of non-supplemented subjects.
Keywords: Folate; 5-Methyltetrahydrofolate; Folic acid; Homocysteine; Tandem mass spectrometry;
Column-switching HPLC–MS/MS analysis of ropivacaine in serum, ultrafiltrate and drainage blood for validating the safety of blood reinfusion by Torben Breindahl; Ole Simonsen; Kirsten Andreasen (76-82).
A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) method, using back-flush column-switching was developed for total drug concentrations of ropivacaine in serum and drainage blood in the measuring range 0.1–10 μg/mL. Samples were diluted with internal standard (2H7-ropivacaine) and extraction buffer, centrifuged and injected directly onto a BioTrap 500 MS extraction column. Using a time programmed six-port valve switch, ropivacaine was back-flushed onto a Zorbax SB-Aq analytical column, gradient eluted and finally detected after electro spray ionisation and multiple reaction monitoring (MRM) of the transitions m/z 275 → m/z 126 and m/z 282 → m/z 133 for ropivacaine and 2H7-ropivacaine, respectively. Accuracy (bias-%) was −1.5 to 5.8% and intermediate precision (C.V.) was 1.4–3.1%. The low sample amount required (10 μL), high specificity and short run time (6 min) makes it very suitable for determination of ropivacaine. Using the same methodology as described above and 200 μL ultrafiltrate, the free drug concentrations of ropivacaine in serum could be precisely determined with a C.V. below 3%. The method was used to investigate the safety of reinfusion of drainage blood after knee and hip arthroplasty when ropivacaine (Naropin®) was used for local analgesia. Data for 30 patients are summarised.
Keywords: Anaesthetics; Local infiltration analgesia; Ropivacaine; Serum; Drainage blood; Reinfusion; Autotransfusion; Liquid chromatography–mass spectrometry; Column-switching;
Supercritical fluid extraction and high performance liquid chromatographic determination of benzopyrans and phloroglucinol derivative in Hypericum polyanthemum by Simone Tasca Cargnin; Jéssica de Matos Nunes; Juliana Schulte Haas; Luís Fernando Baladão; Eduardo Cassel; Rubem Figueiró Vargas; Sandra Beatriz Rech; Gilsane Lino von Poser (83-87).
The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO2) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 °C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 °C and for the high molecular-weight compound, uliginosin B, higher pressures were required.
Keywords: Supercritical extraction; Hypericum; Benzopyrans; Phloroglucinol derivative; Herb processing;
2-Propanol in the mobile phase reduces the time of analysis of CLA isomers by silver ion-HPLC by Katrin Kuhnt; Christian Degen; Gerhard Jahreis (88-91).
Individual isomers of octadecadienoic acid (C18:2) with conjugated double bonds (conjugated linoleic acids; CLA) exert different biological activities. Their distribution in food and tissues differs. Therefore, the separation of the various positional and geometric isomers is important. The time of analysis using silver ion-high performance liquid chromatography can extend up to 90 min. The aim of this study was to reduce this time. The time of analysis reduced from ca. 90 min onto 45 to 35 min, respectively, by the addition of 0.05% or 0.1% (v/v) 2-propanol to the mobile phase [acetonitrile (0.1%; v/v) and diethyl ether (0.5%; v/v) in n-hexane]. There was no effect on resolution of the 17 individual CLA isomers of the CLA mixture. Regarding the lowest coefficient of variation and an adequate baseline separation the use of 0.05% 2-propanol in the mobile phase is recommended, without any disadvantages and adverse effects on the service life of columns. In conclusion, adding 0.05% or 0.1% 2-propanol to the mobile phase shortens the time of analysis of CLA isomers, saves solvents and reduces costs.
Keywords: Conjugated linoleic acids; Silver ion-high performance liquid chromatography; Time of analysis; Geometric isomers;
Quantifying the HIV-1 integrase inhibitor raltegravir in female genital tract secretions using high-performance liquid chromatography with ultraviolet detection by Jasmine A. Talameh; Naser L. Rezk; Angela D.M. Kashuba (92-96).
Understanding the pharmacokinetics of drugs in peripheral body compartments, such as the genital tract, is particularly important in the infectious diseases arena. However, extracting drugs from small volumes of viscous, proteinacious substances like cervicovaginal fluid is particularly challenging. The goal of this study was to develop a method to quantify raltegravir, an HIV-1 integrase inhibitor, in the female genital tract. The method included sample preparation with perchloric acid followed by solid-phase extraction, separation with reverse-phase high-performance liquid chromatography, and detection with an ultraviolet wavelength of 218 nm. The method was linear from 0.05 to 10.0 mg/L, with minimal endogenous interference. The method was accurate (1.2–11.0% deviation) and precise (1.1–12.6% CV) for both within and between-day analyses. The ability to detect raltegravir in the female genital tract is essential for future investigations of raltegravir as an agent for prevention of HIV acquisition, and this method will be used for clinical studies further evaluating pharmacokinetic–pharmacodynamic relationships in this body compartment.
Keywords: Raltegravir; Integrase inhibitor; Female genital tract; Cervicovaginal fluid; Solid-phase extraction; High-performance liquid chromatography;
Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS, and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity by Ursula Lutz; Nataly Bittner; Mike Ufer; Werner K. Lutz (97-101).
Drug–drug and food–drug interactions are often due to an inhibition or induction of drug-metabolizing cytochrome P450 (CYP) enzymes and may result in non-response or adverse reactions. Hence, phenotypic biomarkers of CYP activity appear as useful tools for individualized pharmacotherapy. The metabolic ratio (MR) of the concentration of 6β-hydroxycortisol (6β-OHC) to cortisol (MR 6β-OHC/cortisol) in human urine had been proposed as an endogenous marker for CYP3A activity. Here, we report on the improvement of published LC-MS/MS methods for the simultaneous quantification of cortisol and 6β-OHC, using on-line sample cleanup by column switching and isotope-labeled analogues as internal standards. [2H2]6β-OHC was prepared by incubation of human recombinant CYP3A4 with commercially available [2H2]cortisol. Analytical sensitivity could be increased about 10-fold. The first morning urine of 69 female and 27 male healthy volunteers was analyzed for cortisol and 6β-OHC. Concentrations ranged from 1.0 to 142 and 24 to 670 ng/mL, respectively. Individual MR 6β-OHC/cortisol varied more than 20-fold and we were able to show for the first time for a Caucasian population significantly higher MR values in females as compared to males. This non-invasive biomarker for CYP3A activity lends itself for the study of genetic differences as well as enzyme induction or inhibition in the clinical setting without the need of using a probe drug.
Keywords: Cortisol; CYP3A; Metabolite profiling; Humans; Urine; Phenotyping; Gender;
An LC-MS/MS method for determination of forsythiaside in rat plasma and application to a pharmacokinetic study by Geng-Nan Wang; Rui-Le Pan; Yong-Hong Liao; Ying Chen; Jing-Tian Tang; Qi Chang (102-106).
A highly sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of forsythiaside in rat plasma using epicatechin as internal standard. The analytes were extracted by solid-phase extraction and chromatographied on a C18 column eluted with a gradient mobile phase of acetonitrile and water both containing 0.2% formic acid. The detection was performed by negative ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 623 → 161 and m/z 289 → 109 for forsythiaside and epicatechin, respectively. The assay was linear over the concentration ranges of 2.0–50.0 and 50.0–5000.0 ng/mL with limits of detection and quantification of 0.2 and 1.0 ng/mL, respectively. The precision was <10.8% and the accuracy was >91.9%, and extraction recovery ranged from 81.3% to 85.0%. This method was successfully applied to a pharmacokinetic study of forsythiaside in rats after intravenous (20 mg/kg) and oral (100 mg/kg) administration, and the result showed that the compound was poorly absorbed with an absolute bioavailability being approximately 0.5%.
Keywords: Forsythiaside; LC-MS/MS; Pharmacokinetics; Bioavailability;
Determination of nicotine and cotinine in human serum by means of LC/MS by F. Baumann; R. Regenthal; I.L. Burgos-Guerrero; U. Hegerl; R. Preiss (107-111).
As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.
Keywords: Nicotine; Cotinine; LC/MS; SPE;