Journal of Chromatography B (v.978-979, #C)

An LC–MS/MS method for the determination of five erectile dysfunction drugs and their selected metabolites in hair by Sooyeun Lee; Boyeon Choi; Jihyun Kim; Sanghwan In; Seungkyung Baeck; Seung Min Oh; Kyu Hyuck Chung (1-10).
The abuse of sildenafil and its analogous, accelerated by their inappropriate or illegal distribution, is a serious social issue globally. However, no studies have been conducted to monitor these drugs simultaneously in hair, which can provide valuable information on chronic drug use. In the present study, an LC–MS/MS method was developed for the simultaneous determination in hair of five erectile dysfunction drugs having a high risk for abuse (mirodenafil, sildenafil, tadalafil, udenafil and vardenafil) and their selected metabolites (SK3541, desmethylsildenafil, DA8164 and desethylvardenafil). The novel method was fully validated after optimizing matrix effects and extraction efficiency. The optimized sample preparation included acidic methanol extraction followed by solid phase extraction using C18 mixed mode strong cation exchange polymeric cartridges. The prepared samples were analyzed by LC–MS/MS with electrospray ion source in the positive ionization mode. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. LODs ranged from 0.05 (DA8164) to 1 ng/10 mg hair (tadalafil). LOQs were 1 ng/10 mg hair except for DA8164 and vardenafil, of which they were 2.5 ng/10 mg hair. No significant variations were observed by different sources of matrices in both human and rat hair, except for tadalafil, for which a stable isotope-labeled internal standard was effective. The animal study suggested hair pigmentation was a major factor for the incorporation of the drugs and metabolites into hair. However, a wide variation of the sildenafil-to-desmethylsildenafil ratios was observed in human hair samples. The developed method will be very useful for monitoring the abuse of erectile dysfunction drugs for both legal and public health aspects.
Keywords: Erectile dysfunction drugs; Drug abuse; Hair analysis; Liquid chromatography–tandem mass spectrometry; Matrix effects; Hair pigmentation;

5,7-Dimethoxyflavone (5,7-DMF), a natural flavonoid abundant in many plants, has been reported to have many beneficial pharmacological effects, including strong chemopreventive and chemosensitizing properties, anti-oxidant, cardiovascular protectant, and anti-inflammatory activities. However, to date 5,7-DMF was evaluated mainly in vitro and its pharmacokinetics (PK) in vivo remains largely unknown. In addition, current available quantification methods of 5,7-DMF all lack sufficient sensitivity (lower limit of quantification >800 ng/mL). The purposes of our study are to establish a sensitive quantification method of 5,7-DMF using LC–MS/MS and evaluate the PK profile of 5,7-DMF in mouse. Our method was fully validated and all of the fundamental parameters in method validation, including accuracy, precision, sensitivity, selectivity, recovery and stability were evaluated thoroughly in mouse plasma. The calibration curve covered 2–1000 ng/mL with the lower limit of quantification (LLOQ) of 2 ng/mL. The inter-run and intra-run precision and accuracy were less than 15% of nominal concentrations. The matrix effect and recovery yield were within ±15% of nominal concentrations. In the PK study, 5,7-DMF was detectable in mouse plasma up to 21 h, with a terminal half-life of 11.5 h. The clearance of 5,7-DMF (CL/F) is 22.3 L/h/kg and area under the curve (AUCinf) is 449 h ng/mL. In conclusion, a fast, accurate, sensitive and selective quantification method of 5,7-DMF was established and the developed method was successfully applied to a PK study of 5,7-DMF following oral administration of 5,7-DMF in mice.
Keywords: 5,7-Dimethoxyflavone; LC–MS/MS; Pharmacokinetics; Natural product;

Determination of urinary cortisol, cortisone and 6-sulfatoxymelatonin using dilute and shoot ultra-high pressure liquid chromatography–tandem mass spectrometry by Anna Maria Sniecinska-Cooper; Ajit Jesang Shah; Dagmara Dimitriou; Ray Kruse Iles; Stephen Andrew Butler; Richard Bayford (18-23).
Human sleep is a natural part of every individual's life. Clear relationship between sleep and endocrine system has been already established. In particular, melatonin and cortisol are known to affect and regulate sleep/wake patterns. Here we report the development of an ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of 6-sulfatoxymelatonin (MT6s), cortisol and cortisone in urine. A separate method was developed for measurement of creatinine in urine. These levels were used to normalise the levels of analytes. First void morning urine samples were collected from 24 healthy volunteers. Samples were diluted 1:1 in water prior to injection onto reversed-phase C18 column and analysed using UHPLC–MS/MS method. Linear calibrations were obtained for all analytes with correlation coefficient in the range 0.998–0.999. The observed concentration was found to be in the range 92–105% for cortisol, 92–107% for cortisone and between 93 and 120% for MT6s of the reference levels. The total run time of 6 min with all peaks of interest eluting within 3 min was obtained. This demonstrates the feasibility of utilising the method for large multi-scale studies, where high throughput is required for studying the circadian rhythm of melatonin and cortisol secretion. These hormones play significant role in circadian rhythm and sleep/wake cycle; therefore it is important to monitor the levels of these endocrine markers in individuals suffering from sleep disorders. It is also beneficial with clinical applications to analyse melatonin and cortisol simultaneously in order to assess their interrelationships of these substances, such as their effect on diurnal rhythm and sleep.
Keywords: Cortisol; Cortisone; 6-Sulfatoxymelatonin; UHPLC–MS/MS; Urine; Sleep;

Icaritin (ICT), a bioactive metabolite of prenylflavonoids from genus Epimedium, has displayed potential benefits for the treatment of osteoporosis, prostate cancer, liver cancer, renal cancer and breast cancer. To investigate the quantity of ICT in bones in vivo, a simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed. After a rapid one-step liquid–liquid extraction using ethyl acetate with recovery more than 87.2% at four levels (0.1, 0.2, 8 and 15 ng/mL), ICT and internal standard coumestrol were analyzed on a C18 column using a gradient elution of acetonitrile and water containing ammonium formate and formic acid at a flow rate of 0.3 mL/min. Quantification was performed using selected reaction monitoring mode to monitor precursor-product ion transitions of m/z 367.1 → 297.1 for ICT and of 267.0 → 211.1 for coumestrol in the negative ionization mode. A calibration curve with good linearity (r  > 0.99) within the concentration range of 0.1–20 ng/mL for ICT was obtained with the lower limit of quantification of 0.1 ng/mL. Matrix effect did not interfere with ICT analysis and ICT was stable under three freeze–thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a dynamic distribution of ICT in mouse bone after a single intraperitoneal administration to ICT to mice.
Keywords: Icaritin; UHPLC–MS/MS; Bone;

To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13 ng/ml (signal/noise = 3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area (n  = 3) were 0.03–1.15 and 0.72–6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise = 3), The relative standard deviations for migration time and peak area (n  = 6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.
Keywords: Capillary electrophoresis; Nucleic acid; Field-amplified; Sensitivity;

An ultra high performance liquid chromatography with tandem mass spectrometry (U-HPLC–MS/MS) method was developed for simultaneous determination and pharmacokinetic study of ten active constituents, phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin in rat plasma after oral administration of Yankening Capsule. After mixing with two internal standards tetrahydropalmatine and rutin, plasma samples were pretreated by protein precipitation with anhydrous ethanol–acetonitrile (9:1, v/v). The U-HPLC separation was carried on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 μm) with gradient elution using a mobile phase composed of methanol and water (containing 0.3% formic acid) at a flow rate of 0.3 mL min−1. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive–negative ionization mode. The calibration curves of ten analytes showed good linearity (r  > 0.9979). The lower limits of quantification of phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin were 0.50, 0.50, 0.30, 0.30, 0.30, 10, 3.0, 8.0, 1.0, 8.0 μg L−1, respectively. The relative standard deviation of intra-day precision and inter-day precision were in the range from 1.13% to 5.96% and from 0.65% to 8.85%, respectively. The matrix effects of all analytes were found to be within the acceptable range with a range of 89.99–109.3%. The method is reliable and rapid and has been applied successfully to pharmacokinetic study of the ten active constituents in rat plasma after oral administration of Yankening Capsule.
Keywords: Pharmacokinetics; Active constituents; U-HPLC–MS/MS; Yankening Capsule;

A simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of six components including apigenin, quercetin, apigenin-7-O-β-d-glucoside, quercetin-3-O-β-d-glucoside, 3′-methoxyluteolin-7-O-β-d-glucoside, and tricin-7-O-β-d-glucopyranoside in rat plasma using formononetin as the internal standard (IS). The plasma samples were pretreated by a one-step liquid–liquid extraction with dichloromethane. The chromatographic separation was carried out on a ZORBAX SB-Aq column with a gradient mobile phase consisting of acetonitrile and 2 mM aqueous ammonium acetate. All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring mode. The mass transitions were as follows: m/z 269.1 → 117.2 for apigenin, m/z 301.2 → 151.2 for quercetin, m/z 431.3 → 311.2 for apigenin-7-O-β-d-glucoside, m/z 463.2 → 300.2 for quercetin-3-O-β-d-glucoside, m/z 461.3 → 283.1 for 3′-methoxyluteolin-7-O-β-d-glucoside, m/z 491.3 → 313.1 for tricin-7-O-β-d-glucopyranoside, and m/z 267.2 → 252.2 for IS, respectively. All calibration curves exhibited good linearity with correlation coefficient (r) > 0.995. The intra-day and inter-day precisions (RSD) at three QC levels were both less than 14.0% and the accuracies ranged from 89.8% to 113.8%. The extraction recoveries of six compounds ranged from 82.3% to 92.5%. The validated method was successfully applied to pharmacokinetic study of the six components in male rat plasma after oral administration of Paulownia tomentosa flower extract.
Keywords: LC–MS/MS; Flavonoids; Paulownia tomentosa; Pharmacokinetic study;

The analysis of linear and monomethylalkanes in exhaled breath samples by GC × GC-FID and GC–MS/MS by Alexandra Hengerics Szabó; Peter Podolec; Viktória Ferenczy; Róbert Kubinec; Jaroslav Blaško; Ladislav Soják; Renáta Górová; Gabriela Addová; Ivan Ostrovský; Jozef Višňovský; Václav Bierhanzl; Radomír Čabala; Anton Amann (62-69).
A new arrangement of the INCAT (inside needle capillary adsorption trap) device with Carbopack X and Carboxen 1000 as sorbent materials was applied for sampling, preconcentration and injection of C6 ―C19 n-alkanes and their monomethyl analogs in exhaled breath samples. For the analysis both GC–MS/MS and GC × GC-FID techniques were used. Identification of the analytes was based on standards, measured retention indices and selective SRM transitions of the individual isomers. The GC–MS/MS detection limits were in the range from 2.1 pg for n-tetradecane to 86 pg for 5-methyloctadecane. The GC × GC-FID detection limits ranged from 19 pg for n-dodecane to 110 pg for 3-methyloctane.
Keywords: Exhaled breath; INCAT; Monomethylalkanes; n-Alkanes; Needle trap;

A validated method for quantifying hypoglycin A in whole blood by UHPLC–HRMS/MS by Jérémy Carlier; Jérôme Guitton; Cécile Moreau; Baptiste Boyer; Fabien Bévalot; Laurent Fanton; Jean Habyarimana; Gilbert Gault; Yvan Gaillard (70-77).
Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a nutritious and readily available fruit which is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of several species of maple (Acer spp.) which are suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy in Europe, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning. Analysis was carried out using solid-phase extraction (HILIC cartridges), dansyl derivatization and UHPLC–HRMS/MS detection. The method was validated in whole blood with a detection limit of 0.35 μg/L (range: 0.8–500 μg/L). This is the first method applicable in forensic toxicology for quantifying HGA in whole blood. HGA was quantified in two serum samples from horses suffering from atypical myopathy. The concentrations were 446.9 and 87.8 μg/L. HGA was also quantified in dried arils of unripe ackee fruit (Suriname) and seeds of sycamore maple (Acer pseudoplatanus L.) (France). The concentrations were 7.2 and 0.74 mg/g respectively.
Keywords: Hypoglycin A; Ultra-high performance liquid chromatography–high resolution tandem mass spectrometry (UHPLC–HRMS/MS); Blighia sapida Koenig; Jamaican vomiting sickness; Acer pseudoplatanus L.; Equine atypical myopathy;

Rapid determination of fenoldopam in human plasma by UPLC–MS/MS for pharmacokinetic analysis in patients by Xipei Wang; Zhijie Zheng; Guodong He; Liping Mai; Zhiling Zhou; Shilong Zhong; Qiuxiong Lin; Zhixin Shan; Chunyu Deng; Min Yang; Xiyong Yu (78-82).
We developed and validated a rapid, selective, and sensitive ultra-performance liquid-chromatography mass-spectrometry (UPLC–MS/MS) method for quantifying fenoldopam in human plasma for pharmacokinetic studies. Fenoldopam and the internal-standard (IS), oxazepam, were isolated from human plasma by liquid–liquid extraction using ethyl acetate after alkalization, and were separated on a 2.1 × 100 mm Acquity UPLC HSS T3 C18 column (inside diameter, 1.8 μm) using a mobile phase of water (0.05% formic acid) and acetonitrile gradient elution. The fenoldopam and IS were eluted at 1.07 and 2.32 min, respectively. Quantification was performed using positive-ion electrospray-ionization (ESI), and the fenoldopam and IS responses were optimized at the m/z 306.16 → 107.10 and m/z 287.1 → 241.01 transitions, respectively. The assay was validated over the linear range of 0.1–40 ng/mL fenoldopam with intra- and interassay precision <13.21%. The matrix effect of normal and hemolyzed plasma was 94.9–101.6%. Fenoldopam was stable for ≥34 days at −70 °C in normal and hemolyzed plasma containing ascorbic acid as a stabilizer. This method can be successfully applied in pharmacokinetic studies of fenoldopam in hypertensive patients.
Keywords: Fenoldopam; UPLC–MS/MS; Human plasma; Pharmacokinetics; Hemolyzed plasma;

LC–MS analysis of the plasma metabolome—A novel sample preparation strategy by Kasper Skov; Niels Hadrup; Jørn Smedsgaard; Henrik Frandsen (83-88).
Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC–MS based analysis of the plasma metabolome is a challenge as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry.We have compared two different sample preparation techniques prior to LC-qTOF analysis of plasma samples: the first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples: a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples revealed 1792 molecular features from the protein precipitation procedure. The protein precipitation followed by solid phase extraction procedure with three sub-samples gave a total of 4234 molecular features. This suggests that sub-sampling into polar, lipid and phospholipid fractions enables extraction of more metabolomic information as compared to protein precipitation alone. Chromatography showed good separation of the metabolites with little retention time drift (<1 s) and a mass accuracy below 3 ppm was observed. The performance of the method was investigated using plasma samples from rats administered the environmental pollutant perfluorononanoic acid.
Keywords: Metabolomics; LC–MS; Plasma samples; Solid phase extraction;

Ultrafast quantification of β-lactam antibiotics in human plasma using UPLC–MS/MS by Mieke Carlier; Veronique Stove; Jan J. De Waele; Alain G. Verstraete (89-94).
There is an increasing interest in monitoring plasma concentrations of β-lactam antibiotics. The objective of this work was to develop and validate a fast ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC–MS/MS) for simultaneous quantification of amoxicillin, cefuroxime, ceftazidime, meropenem and piperacillin with minimal turn around time. Sample clean-up included protein precipitation with acetonitrile containing 5 deuterated internal standards, and subsequent dilution of the supernatant with water after centrifugation. Runtime was only 2.5 min. Chromatographic separation was performed on a Waters Acquity UPLC system using a BEH C18 column (1.7 μm, 100 mm × 2.1 mm) applying a binary gradient elution of water and methanol both containing 0.1% formic acid and 2 mmol/L ammonium acetate on a Water TQD instrument in MRM mode. All compounds were detected in electrospray positive ion mode and could be quantified between 1 and 100 mg/L for amoxicillin and cefuroxime, between 0.5 and 80 mg/L for meropenem and ceftazidime, and between 1 and 150 mg/L for piperacillin. The method was validated in terms of precision, accuracy, linearity, matrix effect and recovery and has been compared to a previously published UPLC–MS/MS method.
Keywords: Beta-lactam antibiotics; Therapeutic drug monitoring; UPLC–MS/MS;

Polar lipids (PLs) are a significant functional component of milk that are difficult to quantitate. A simple method for comprehensive identification and quantitative analysis of all essential PL species using bovine milk is described. The lipid fraction was extracted by a mix of chloroform and methanol and the extract was directly used for PL identification and quantification. PLs were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyser in positive mode. The structure of PLs was established or confirmed by tandem MS in both positive and negative modes. The method is sensitive (with a LOD for all PL classes ≤0.1 ng) and reproducible, enabling simultaneous quantification of 70 PL species within a run of 45 min. Application of this method to the quantification of PLs in 32 bovine milk samples revealed the relative abundance of different PL classes, significant variation of PL content between individual samples and the correlation between the major PL classes. The method provides a tool for investigating the variation and metabolism of important PL components in bovine and human milk and in diverse mammalian species.
Keywords: Phospholipids; Sphingolipids; Hydrophilic interaction liquid chromatography; Milk; LC–MS;

Multi-residues determination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method by Mamdouh R. Rezk; Safa’a M. Riad; Fatma I. Khattab; Hoda M. Marzouk (103-110).
A rapid, simple, sensitive and specific LC–MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC–MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50 mm × 3 mm, 2.7 μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4 mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1–100 ng/g for both CPX and TMP, 0.5–100 ng/g for SDM and 1–50 ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5 min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms.
Keywords: Antimicrobials; Aquaculture; Fish tissues; Sample extraction; LC–MS/MS;

Preparative separation of echinocandin B from Aspergillus nidulans broth using macroporous resin adsorption chromatography by Shu-Ping Zou; Miao Liu; Qiu-Liang Wang; Yan Xiong; Kun Niu; Yu-Guo Zheng; Yin-Chu Shen (111-117).
Echinocandin B (ECB), an echinocandin type of lipopeptide antibiotic produced by Aspergillus nidulans, is a precursor for the synthesis of novel anti-fungal drug – anidulafungin. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography was established for ECB purification from Aspergillus nidulans CCTCC M 2010275 fermentation broth. Among nine macroporous resin adsorbents tested, the non-polar resin HP-20 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Langmuir equation, and the adsorption kinetic followed the pseudo-second order model. The separation parameters of ECB from broth were optimised by dynamic adsorption/desorption experiments with the column packed with HP-20 resin. Under optimal conditions, the purity increased by 3.8-fold from 23.2% in broth to 88.5% in eluent with 87.1% recovery yield by a one-step treatment. Our study provided a one-step and effective method for large-scale production of ECB, and offered references for separating other echinocandins from broth.
Keywords: Aspergillus nidulans; Echinocandin B; Broth; Macroporous resin; Adsorption chromatography;

Fast separation and quantification of steroid hormones Δ4- and Δ7-dafachronic acid in Caenorhabditis elegans by Michael Witting; Hans-Christian Rudloff; Manjunatha Thondamal; Hugo Aguilaniu; Philippe Schmitt-Kopplin (118-121).
Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones Δ4- and Δ7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. Δ4- and Δ7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3 μm core–shell particle in less than 10 min together with a simple MeOH extraction. Using this method we were able to independently quantify Δ4- and Δ7-DA in C. elegans independently from each other and limits of detection of about 5 ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation.
Keywords: Dafachronic acid; Isomer separation; Caenorhabditis elegans; Sub-2 μm core–shell particles;

A simple, rapid and solvent-free multi-residue method has been developed and applied to confirm and quantify a series of volatile compounds in five cherry wines by gas chromatography coupled with mass spectrometry (GC–MS). Four parameters (e.g., coating material of fiber, temperature and time of extraction, and addition of sodium chloride in the solution) of headspace solid-phase micro-extraction (HS-SPME) were optimized, resulting in the best extraction condition including 50/30 μm DVB/CAR/PDMS fiber, 45 min and 50 °C of SPME, and 2 g of sodium chloride addition in the wine during the extraction. The SPME had LODs and LOQs ranging from 0.03 to 7.27 μg L−1 and 0.10 to 24.24 μg L−1 for analytic compounds, respectively. Repeatability and reproducibility values were all below 19.8%, with mean values of 12.7% and 10.5%, respectively. Regression coefficients (R 2) of detective linearity of the standard curves was higher than 0.9852. Moreover, relative recoveries of analytical targets were achieved in a range of 60.7–125.6% with good relative standard deviation values (≤20.6%). In addition, a principal component analysis (PCA) was used to analyze the aroma profiles of the wines, which indicated that five samples were distinctly divided into two groups based on their different geographical origins and volatile compounds.
Keywords: Cherry wine; SPME; Gas chromatography–mass spectrometry; Volatiles; PCA;

Spermine and spermidine are multiple-nitrogen compounds found in many foods. Both compounds are essential for cell growth and human health. This study established a simple and fast method of detecting spermine and spermidine in food samples by matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI–TOF MS). After a simple sample preparation procedure, spermine and spermidine were directly detected by MALDI–TOF MS with no additional purification procedure. The calibration curves for spermine and spermidine ranged from 0.1 to 10 μg/mL. In intra- and inter-batch assays of three different concentrations of spermine and spermidine, all relative standard deviations and relative errors were below 18.9%. These experimental results confirmed the practicability and effectiveness of the proposed MALDI–TOF MS method for fast determination of spermine and spermidine in food samples. Furthermore, since spermine and spermidine have important roles in apoptosis, up-regulation and down-regulation of spermine and spermidine during apoptosis were analyzed. After treating NRK-52E cells with spermine and spermidine, the cells were lysed, and cell proteins were collected, and digested. Apoptosis-related proteins were then identified by tandem MS.
Keywords: Spermine; Spermidine; Apoptosis; Food; MALDI–TOF MS; NanoUPLC–MS/MS;

A sensitive and rapid method based on formate-adduct ion detection was developed and fully validated for digoxin determination in rat plasma. For LC/MS/MS detection with formate-adducts as precursor ions, transitions of m/z 825.5 → 779.9 for digoxin and m/z 809.5 → 763.4 for the internal standard (digitoxin) were monitored in negative mode. To investigate the impact of formic acid on the mass response and method sensitivity, a formic acid concentration range of 0–0.1% (0, 0.0005%, 0.002%, 0.01%, 0.1%, v/v) was evaluated. A concentration of 0.002% gave the highest sensitivity, which was 16- to 18-fold higher than deprotonated ions, and was designated as the contribution giving the strongest ionization enhancement and adduction. A number of parameters were then varied in order to optimize the method, and a limit of quantitation (LOQ) at 0.2 ng/mL was reached with an injection volume of 5 μL, a total run time of 3 min, and 0.1 mL of rat plasma. A calibration curve was plotted over the range 0.2–50 ng/mL (R 2  = 0.9998), and the method was successfully applied to study pharmacokinetics in rat following a single oral administration of digoxin (0.05 mg/kg). Four additional steroid saponins (digitoxin, deslanoside, ginsenoside Rg1 and Rb1) were investigated to assess the impact of formic acid on the mass response of steroid saponins. Compounds with a conjugated lactonic ring in their structures such as digoxin, digitoxin and deslanoside tended to form stable formate-adduct ions more easily. The LC/MS/MS method developed here is therefore well suited for the quantification of steroid saponins that are difficult to deprotonate using other MS approaches.
Keywords: Digoxin; LC/MS/MS; Formate-adduct; Sensitivity; Rat plasma;

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.
Keywords: Affinity chromatography; Arginine monolith; Composite Central Face design; Design of experiments; HPV-16 E6/E7 vaccine; Supercoiled plasmid DNA;

A novel, specific and sensitive bioanalytical method has been developed for the determination of sucrose octasulfate (SOS) in dog plasma and urine using ion-pair reversed-phase ultraperformance liquid chromatography coupled with electrospray triple quadruple mass spectrometry (IPRP-UPLC ESI MS/MS). 13C-labeled sucrose octasulfate-13C12 sodium salt is used as the internal standard. 200 μL of plasma or serum sample is extracted using weak anion exchange solid phase cartridge. In this method, a polar amide column is employed for the liquid chromatograph (LC) separation while the diethylamine and formic acid buffer is used as the ion-pairing reagent. The low limitation of quantitation of sucrose octasulfate is 0.20 ng on the column with a signal to noise ratio larger than 50. Parameters such as linearity, accuracy and precision have been validated in full compliance with the FDA guidelines for the bioanalytical method development and validation. A linear regression model fit the calibration curve very well with R  > 0.99. The bias and coefficient of variation of all levels of QCs are within the range of 15%. The selectivity, matrix effect and stabilities of analytes in solution and matrix have also been evaluated and the results met the acceptance criteria according to the guidelines. Based on these results, the method has qualified to analyze sucrose octasulfate in dog plasma for clinic research. This method has been applied to 1000 preclinical samples.
Keywords: Sucrose octasulfate; Ion pairing; UPLC–MS/MS;

Simultaneous quantification of naproxcinod and its active metabolite naproxen in rat plasma using LC–MS/MS: Application to a pharmacokinetic study by Xiaowei Shi; Weiding Shang; Shuang Wang; Na Xue; Yanxia Hao; Yabo Wang; Mengmeng Sun; Yumin Du; Deying Cao; Kai Zhang; Qingwen Shi (157-162).
In this study, a liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine naproxcinod and naproxen concentrations in rat plasma for the first time. Plasma samples were prepared by simple one-step extraction with methanol for protein precipitation using only 50 μL plasma. Separation was performed on a Synergi Fusion-RP C18 column with a run time of 4 min. Naproxcinod, naproxen and internal standard concentrations were detected in the positive ion mode using multiple reaction monitoring (MRM) of the transitions at m/z 348.2 → 302.2, 231.1 → 185.1 and 271.2 → 203.1, respectively. The calibration curves were linear, with all correlation coefficients being ≥0.9952, in the range of 1.00–400 ng/mL for naproxcinod and 20.0–8000 ng/mL for naproxen. Their accuracy was in the range of −8.1% to 8.7%, and the intra- and inter-day variations were ≤4.53%. The mean extraction recovery of all analytes was more than 93.1% efficient. Stability testing showed that naproxcinod and naproxen remained stable during the whole analytical procedure. After validation, the method was successfully applied to a pharmacokinetic study of naproxcinod and naproxen in rats. The AUC0–∞ of naproxen was 74.6 times larger than that of naproxcinod, which indicated that naproxcinod was rapidly metabolized into naproxen in rats.
Keywords: Naproxcinod; Naproxen; LC–MS/MS; Pharmacokinetic study;

Measurement of intracellular ribavirin mono-, di- and triphosphate using solid phase extraction and LC–MS/MS quantification by Leah C. Jimmerson; Michelle L. Ray; Lane R. Bushman; Peter L. Anderson; Brandon Klein; Joseph E. Rower; Jia-Hua Zheng; Jennifer J. Kiser (163-172).
Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC–MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC–MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection.
Keywords: Ribavirin triphosphate; Intracellular pharmacology; Analytical methods; LC–MS/MS; Dried blood spots; Clinical pharmacology;

To study the systemic exposure of tazarotene formulation after topical administration, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of tazarotene and tazarotenic acid in minipig plasma. Similar extraction recoveries for both analytes were obtained after the plasma samples were acidified by glacial acetic acid (5%) and extracted by ethyl ether–cyclohexane (4:1, v/v). Separation of the analytes was achieved within a short time by the addition of 0.1% formic acid to the mobile phase. Gradient elution was used to avoid the matrix effect. The method was linear over the concentration range of 10–600 pg/mL for both analytes. The data of intra- and inter-run precision and accuracy were lower than 5.2%, 7.3% and 7.3% for both analytes. The developed method can be applied to investigate the transdermal pharmacokinetics and the systemic exposure of tazarotene formulation after topical administration.
Keywords: Tazarotene; Tazarotenic acid; LC–MS/MS; Systemic exposure; Transdermal administration;