Journal of Chromatography B (v.1022, #C)

Determination of albendazole sulfoxide in human plasma by using liquid chromatography–tandem mass spectrometry by Nihal Saraner; Güler Yağmur Özkan; Berrak Güney; Erkin Alkan; Nihan Burul-Bozkurt; Onursal Sağlam; Ezgi Fikirdeşici; Mevlüt Yıldırım (1-5).
A rapid, simple and sensitive method was developed and validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for determination of albendazole sulfoxide (ABZOX) in human plasma. The plasma samples were extracted by protein precipitation using albendazole sulfoxide-d3 as internal standard (IS). The chromatographic separation was performed on Waters Xbridge C18Column (100 × 4.6 mm, 3.5 μm) with a mobile phase consisting of ammonia solution, water and methanol at a flow rate of 0.70 mL/min. ABZOX was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 3–1500 ng/mL for ABZOX. This method was successfully applied to the bioequivalence study in human plasma samples.
Keywords: Albendazole sulfoxide; Albendazole; Albendazole sulfoxide-d3; Human plasma; LC–MS/MS;

7′(Z)-(8″S, 8‴S)-epi-Salvianolic acid E (compound 1) and (7′R, 8′R, 8″S, 8‴S)-epi-salvianolic acid B (compound 2), two novel analogs of salvianolic acid B (Sal B), have been recently isolated from Salvianolic acid for injection. They both show powerful antioxidant effects, including inducing NQO1 activity and scavenging DPPH free radical, and potential protecting effects for cerebral ischemia. However, no reports have been described the pharmacokinetic study of them. In this study, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for the determination of compound 1, compound 2 and Sal B in rat plasma, respectively. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters Acquity UPLC® HSS T3 column (1.7 μm particles, 2.1 mm i.d. × 100 mm) with the mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B) (65:35, v/v). Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization (ESI) by multiple reaction monitoring (MRM) in the negative ion mode. Monitored transitions were set at m/z 717.0 → 519.0, 717.1 → 519.1, 717.2 → 518.9 and 320.9 → 152.1 for compound 1, compound 2, Sal B and chloramphenicol (internal standard, IS), respectively. Linear calibration curves were acquired over the concentration range of 2.0–1000 ng/mL for the three analytes in rat plasma. The extraction recoveries, matrix effects, intra- and inter-day precisions and accuracies of the three analytes were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of compound 1, compound 2 and Sal B after intravenous administration of 6.0 mg/kg in rats, respectively. The results indicated that compound 1 and compound 2 were both eliminated more slowly than Sal B. Exposure levels of both compound 1 and Sal B were higher than compound 2 in the same dosage range. This study provided critical reference for the pharmacokinetic study of compound 1 and compound 2.
Keywords: 7′(Z)-(8″S, 8‴S)-epi-Salvianolic acid E; (7′R, 8′R, 8″S, 8‴S)-epi-Salvianolic acid B; Salvianolic acid B; UFLC-MS/MS; Pharmacokinetics;

Anticancer 20(R)-dammarane-3β,12β,20,25-tetrol-loaded polymeric micelles: Preparation, quantification and pharmacokinetics by Junxian Yu; Zhe Li; Wei Wang; Yang Zhang; Dandan Li; Yi Liu; Su Shen; Ruiwen Zhang (13-20).
Polymeric micelles are effective drug-loading sites and often used to formulate poorly water-soluble agents. In the present study, the amphiphilic copolymer methoxy-capped poly(ethyleneglycol)-block-poly(Ɛ-caprolactone) (mPEG-b-PCL) was successfully developed for the delivery of 20(R)-dammarane-3β,12β,20,25-tetrol (25-OH-PPD), a natural anticancer product from Panax notoginseng. The 25-OH-PPD-loaded micelles were characterized by morphological observation and thermodynamic stability testing. The concentrations of 25-OH-PPD was determined by HPLC–MS/MS. The optimum MRM transition of 25-OH-PPD was selected at m/z 479.4.0 → 461.4. The chromatographic separation was achieved on a SB-C18 column (1.8 μm, 2.1 × 50 mm) with an optimized gradient mobile phase system. The extraction recoveries of plasma and various tissue homogenates were within the range of 81.1%–110.4% and the matrix effects ranged from 81.9% to 106.7%. The intra- and inter- day precision values (RSD%) were less than 12.0%, with accuracies ranging from 85.2% to 114.2%. In addition, 25-OH-PPD was found to be stable in different biological matrix after three freeze-thaw cycles, at room temperature and at −70 °C for 4 weeks. The pharmacokinetics of 25-OH-PPD-loaded micelles was evaluated in rats. The micelles appeared as transparent liquid, stable and uniform spheres with an average particle size of 35.4 ± 4.2 nm. The maximum concentration of 25-OH-PPD in micelles was much lower than in free drug preparation. However, the drug in the micelles was released steadily, with a t1/2 of 9.1 ± 4.0 h, significantly longer than in free drug (3.3 ± 1.4 h). However, the drug concentrations in tissues after the micelle administration were lower than the levels after administration of the free drugs. In summary, the micelles were characterized by long circulation and sustained release, with an ability to avoid uptake by the reticuloendothelial system, providing a promising approach to deliver intravenous 25-OH-PPD for therapy.
Keywords: 20(R)-dammarane-3β,12β,20,25-tetrol; Polymeric micelles; LC-ESI–MS/MS; Pharmacokinetics;

The detection of drug metabolites, particularly for minor metabolites, continues to be a challenge owing to the complexity of biological samples. Imperatorin is an active natural furocoumarin ingredient originating from many traditional Chinese herbal medicines. In the present study, the metabolites of imperatorin after oral administration were qualitatively investigated, and possible metabolic pathways of it were subsequently proposed. Bile samples were collected after oral administration and pretreated by the application of Waters Ostro. The QTOF-MS/MS data was acquired using ultra high performance liquid chromatography coupled to quadrupole time flight spectrometry (UPLC-QTOF-MS). Based on this analytical strategy, 32 metabolites (23 phase I and 9 phase II metabolites) were identified in rat bile. The results demonstrated that C5H8 could be easily eliminated from imperatorin forming the metabolite M1. It also indicated that imperatorin and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. This is the first study of imperatorin metabolism in bile samples. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of other linear-type furocoumarins.
Keywords: Imperatorin; Linear-type furocoumarins; Rat bile; Metabolite identification; UPLC-QTOF-MS/MS;

Sensitive analysis and pharmacokinetic study of the isomers paeoniflorin and albiflorin after oral administration of Total Glucosides Of White Paeony Capsule in rats by Fei Fei; Hailing Yang; Ying Peng; Pei Wang; Shuyao Wang; Yuqing Zhao; Jingqiu Huang; Xiaoyi Yu; Siqi Feng; Runbin Sun; Na Yang; Hongbo Wang; Jiye Aa; Guangji Wang (30-37).
A sensitive and reliable method using liquid chromatography tandem mass spectrometry (LC–MS/MS) was established for the simultaneous assay of paeoniflorin and albiflorin in bio-samples of rats after liquid–liquid extraction with ethylacetate. For the first time, the developed method was validated and successfully applied to the pharmacokinetics study of paeoniflorin and albiflorin after oral administration of Total Glucosides Of White Paeony Capsule (TGP). Relative to the intravenous injection, the absolute bio-availabilities of paeoniflorin and albiflorin were 2.8 and 1.7%, while their excretion in feces was 43.06 and 40.87%, respectively. Both paeoniflorin and albiflorin showed dose-dependent exposure in plasma, with a half-life of approximately 1.8 h. No significant differences were observed between a single equal dose of paeoniflorin or albiflorin and that of TGP for the pharmacokinetic parameters, including AUC, T1/2 and Cmax. Paeoniflorin and albiflorin were exposed at high levels in immune relevant organ/tissues, such as the spleen, thymus and bone, which could facilitate immuno-regulatory activities.
Keywords: LC–MS/MS; Albiflorin; Paeoniflorin; Pharmacokinetics; Tissue distribution;

Simultaneous quantitative determination of 5-aza-2′-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors by Nicole M. Anders; Jianyong Liu; Teresia Wanjiku; Hugh Giovinazzo; Jianya Zhou; Ajay Vaghasia; William G. Nelson; Srinivasan Yegnasubramanian; Michelle A. Rudek (38-45).
The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2′-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2′-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2′-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC–MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100 mm × 2.1 mm, 5 μm) and isocratic elution with a 10 mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2′-deoxycytidine, 2′-deoxycytidine, and 5-methyl-2′-deoxycytidine. The assay range was 2–400 ng/mL for 5-aza-2′-deoxycytidine, 50–10,000 ng/mL for 2′-deoxycytidine, and was 5–1000 ng/mL for 5-methyl-2′-deoxycytidine. The assay proved to be accurate (93.0–102.2%) and precise (CV ≤ 6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at −70 °C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2′-deoxycytidine and 5-methyl-2′-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.
Keywords: 5-Aza-2′-deoxycytidine; Decitabine; DNA methyltransferase inhibitor; LC/MS/MS; Genomic DNA; Global DNA demethylation; 5-Methyl-2′-deoxycytidine; 5-Methylcytosine;

2′,3′,5′-Tri-O-acetyl-N6-(3-hydroxylaniline)adenosine (IMM-H007, once called WS070117) is being developed as a novel anti-hyperlipidemia agent for its high efficacy and low toxicity. In this study, a sensitive and specific liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was established for the simultaneous quantification of IMM-H007 and its two major metabolites (3S,4R,5R)-2-(hydroxymethyl)-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahdrofuran-3,4-diol (M1) and ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (MP) in hamster blood. An analogue of IMM-H007, WS070119 was used as the internal standard. Blood samples were prepared by a simple protein precipitation with acetonitrile. The chromatographic separation was performed on a ReproSil-Pur 120C18 column (3 μm, 2 mm × 100 mm) with a gradient mobile phase of methanol/water containing 0.1% formic acid (v/v) in a flow rate of 0.2 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) in positive ion selective reaction monitoring (SRM) mode. The monitored transitions were 486.2 → 228.1 for IMM-H007, 360.0 → 228.0 for M1, 440.0 → 228.0 for MP and 374.1 → 242.0 for the internal standard, respectively. Satisfactory linearity was obtained for the analytes over the range of 1–500 ng/mL for IMM-H007, 2–1000 ng/mL for M1 and 10–5000 ng/mL for MP. The lower limits of the quantification (LLOQs) were 1 ng/mL for IMM-H007, 2 ng/mL for M1 and 10 ng/mL for MP. The intra-day and inter-day precisions (RSD, %) of the analytes were within 14.2%, and the accuracy (RE, %) ranged from −9.4% to 10.7%. The average recoveries of the analytes were more than 80.0%. The analytes were proved to be stable during given storage, preparation, and analytic procedures. The method was successfully applied to the pharmacokinetic study in hamsters after oral administration of IMM-H007.
Keywords: O2′,O3′,O5′-Tri-acetyl-N6-(3-hydroxylaniline)adenosine; Metabolites; Quantification; LC–MS/MS; Pharmacokinetics;

Detection and characterization of clostebol sulfate metabolites in Caucasian population by Georgina Balcells; Oscar J. Pozo; Lorena Garrostas; Argitxu Esquivel; Xavier Matabosch; Aristotelis Kotronoulas; Jesús Joglar; Rosa Ventura (54-63).
Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC–MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3 g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC–MS/MS and GC–MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC–MS/MS screening methods base on the direct detection of phase II metabolites.
Keywords: Clostebol; Sulfate metabolites; LC–MS/MS; Doping analysis; Anabolic steroid;

Quantification of brown dog tick repellents, 2-hexanone and benzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris by Jaires Gomes de Oliveira Filho; André Lucio Franceschini Sarria; Lorena Lopes Ferreira; John C. Caulfield; Stephen J. Powers; John A. Pickett; Adalberto A. Pérez de León; Michael A. Birkett; Lígia Miranda Ferreira Borges (64-69).
We have recently shown that repellency of the tick Rhipicephalus sanguineus sensu lato by the tick resistant dog breed, the beagle, is mediated by volatile organic compounds (VOCs) 2-hexanone and benzaldehyde present in beagle odour. Ectoparasite location of animal hosts is affected by variation in these odour components and their ratios. The aim of this study was to quantify the release rate, and the ratio, of 2-hexanone and benzaldehyde from beagles. The odour of three beagles was collected, for four days, over one week (day 0, day 1, day 4 and day 7). The compounds were identified using coupled high-resolution gas chromatography–mass spectrometry (GC–MS), and authentic standards of compounds were used to generate external calibration curves for quantification. Both compounds were found in all dogs on all days. The amount of benzaldehyde was always higher than that of 2-hexanone and so their ratio varied from unity, on average (over time) being 3.128 ± 0.365, 1.902 ± 0.390, 1.670 ± 0.671 ng mL−1 for beagle 1, 2 and 3, respectively. There was no significant (p < 0.05, F-test) effect of time. The overall mean was 2.233 ± 0.387 ng mL−1. These results further previous findings by documenting the presence of 2-hexanone and benzaldehyde in beagle odour samples covering a 7-day period. This knowledge enables development of repellents to protect dogs from R. sanguineus s. l. infestation.
Keywords: 2-Hexanone; Benzaldehyde; VOC’s; Rhipicephalus sanguineus sensu lato; Non-host; GC–MS;

Bromophenols (BrPs), as the metabolites of PBDEs, would be the potential exposure markers for human biomonitoring (HB) of PBDEs in urine. An analytical method using solid-phrase extraction (SPE) and gas chromatography coupled to tandem mass spectrometer (GC–MS/MS) was developed and validated for simultaneous determination of nineteen BrPs in human urine. The method detection limits (MDLs) were below 23 pg mL−1, with recovery ranged from 63% to 133% and inter-day repeatability ranged from 3% to 11% for the majority of target analytes. This method was applied in a pilot study and 2-Bromophenol (2-BrP), 4-Bromophenol (4-BrP), 2,4-Dibromophenol (2,4-DBP) and 2,4,6-Tribromophenol (2,4,6-TBP) as the predominant analytes were detected in human urine samples collected from the general population. Among the four detected analytes, 2-BrP and 4-BrP as the mono-brominated BrP congeners were firstly reported. To our knowledge, it is the first study covering all BrP congeners (from mono-brominated to penta-brominated, totally 19 congeners) in human urine. Therefore, this study is very useful for profiling urinary BrPs and discovering potential relationship between urinary BrPs and human internal exposure to PBDEs. The mechanism of fragmentation pathway of silanized BrPs was firstly illustrated in this study.
Keywords: Bromophenols; Human urine; PBDEs; Human biomonitoring; GC–MS/MS; Fragmentation pathway;

In order to rapidly and accurately screen compounds present in Chinese herbal medicines for α-glucosidase inhibition activity, we have developed a screening strategy which couples the immobilized enzymes with HPLC analysis. First, the core-shell PMMA/CS nanoparticle carrier was prepared by graft polymerization method. Subsequently, α-glucosidase was loaded on the nanoparticles to prepare the immobilized enzyme and construct a screening model. Secondly, the morphology and structure of the core-shell PMMA/CS material were characterized by transmission electron microscopy (TEM) and infrared spectroscopy (IR). The results indicate that the size distribution was uniform for the α-glucosidase-immobilized nanoparticles, with an average particle size of about 239 nm. Finally, the α-glucosidase-immobilized nanoparticles were used to screen α-glucosidase inhibitors from extracts of traditional Chinese medicine and from a mixture of pharmacologically active compounds. Extracts of traditional Chinese medicines were analyzed by HPLC method before and after incubation with the immobilized enzyme nanoparticles. Using this method, magnoflorine was selectively separated from methanol extracts of Magnoliae Officinalis Cortex and genistein was selectively separated from a mixture of isoflavone compounds. Both the two compounds have previously been reported to have α-glucosidase inhibitory activity. These results demonstrate that this screening strategy represents a rapid and highly efficient method for the identification of α-glucosidase inhibitors in complex extracts of Chinese herbal medicines.
Keywords: Immobilized enzyme; Core-shell nanoparticle; α-Glucosidase; Inhibitors; Screening model;

Development and validation of a selective HPLC-UV method for thymol determination in skin permeation experiments by Tamara Angelo; Felipe Q. Pires; Guilherme M. Gelfuso; Joyce K.R. da Silva; Tais Gratieri; Marcílio S.S. Cunha-Filho (81-86).
Thymol is a natural monoterpene, whose antioxidant and antimicrobial properties suggest a potential use in topical formulations. A simple, precise and selective HPLC method for thymol determination in skin penetration studies was developed and validated in this paper. Separation was achieved with a RP-C18 column, mobile phase comprised of acetonitrile:water (35:65 v/v), flow rate of 1.5 mL/min, oven temperature at 40 °C, injection volume of 30 μL and UV detection at 278 nm. The validation procedure certified the method was selective for thymol determination even when extracted from skin matrix extracts. It was also linear in a range from 0.5 to 15.0 μg/mL, robust, precise and accurate, with recovery rates from the skin layers higher than 90%. Limits of detection and quantification were 0.05 and 0.14 μg/mL, respectively. The method showed, therefore, to be adequate for use in further skin permeation studies employing thymol topical formulations.
Keywords: HPLC method; Skin; Thymol; Topical formulation; Validation;

Production of alpha-amylase from Aspergillus oryzae for several industrial applications in a single step by María C. Porfirif; Esteban J. Milatich; Beatriz M. Farruggia; Diana Romanini (87-92).
A one-step method as a strategy of alpha-amylase concentration and purification was developed in this work. This methodology requires the use of a very low concentration of biodegradable polyelectrolyte (Eudragit® E-PO) and represents a low cost, fast, easy to scale up and non-polluting technology. Besides, this methodology allows recycling the polymer after precipitation.The formation of reversible soluble/insoluble complexes between alpha-amylase and the polymer Eudragit® E-PO was studied, and their precipitation in selected conditions was applied with bioseparation purposes. Turbidimetric assays allowed to determine the pH range where the complexes are insoluble (4.50-7.00); pH 5.50 yielded the highest turbidity of the system. The presence of NaCl (0.05 M) in the medium totally dissociates the protein-polymer complexes.When the adequate concentration of polymer was added under these conditions to a liquid culture of Aspergillus oryzae, purification factors of alpha-amylase up to 7.43 and recoveries of 88% were obtained in a simple step without previous clarification. These results demonstrate that this methodology is suitable for the concentration and production of alpha-amylase from this source and could be applied at the beginning of downstream processing.
Keywords: Alpha-amylase; Intelligent polymer; Bioseparation; Enzymes; Aspergillus oryzae;

In this study, a new analytical method was developed and validated for the simultaneous analysis of β-blockers (metoprolol, propranolol, carvedilol) and their metabolites (5′-hydroxycarvedilol, O-desmethylcarvedilol, α‐hydroxymetoprolol, O-desmethylmetoprolol, 5-hydroxypropranolol) in human urine. A salting-out assisted liquid-liquid extraction (SALLE) procedure was used for sample preparation. Several parameters affecting the extraction efficiency and method sensitivity including the type and volume of the extraction solvent, the type and quantity of the inorganic salt, extraction time and sample pH were investigated. Hydrophilic interaction liquid chromatography-ultraviolet detection (HILIC-UV) was used for the determination of all analytes. During method development, the effects of mobile phase components (type, pH, concentration of salt, organic modifier type and content, flow rate, column temperature) on the retention and separation of β-blockers and metabolites on the five different HILIC columns were examined. The method was linear for concentrations ranging from 0.1 to 8.0 μg/mL, with determination coefficients higher than 0.993 for all analytes. The limits of quantification were in the range from 0.1 to 0.2 μg/mL. Intra- and inter-day precision ranged from 0.1 to 8.9%, and accuracy was within ± 13% interval for all analytes. Under the optimized conditions, extraction efficiency was greater than 83.4% for determined compounds. The validated method was then applied to the measurement of β-blockers and their metabolites in human urine samples.
Keywords: HILIC; SALLE; β-Blockers; Metabolites;

An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus by Weilu Zhou; Weijun Kong; Xiaowen Dou; Ming Zhao; Zhen Ouyang; Meihua Yang (102-108).
An aptamer based lateral flow strip based on competitive format was developed for on-site rapid detection of ochratoxin A (OTA) in Astragalus membranaceus. Some crucial parameters that might influence the sensitive detection, such as the characterization of the colloidal gold, size and shape of gold nanoparticles (AuNPs), amount of AuNPs-aptamer conjugate, migration rate and the addition amount of methanol, were investigated to provide the optimum assay performance. To perform the test, 1 g sample was extracted with 2.5 mL of methanol-water (80:20, v/v) and diluted by 4-fold running buffer to eliminate the matrix and methanol interferences. Under optimized conditions, the aptamer-based assay showed a visual limit of detection (LOD) of 1 ng mL−1, and with no significant cross-reactivity with several homologous toxins. The whole detection could be completed within 15 min without special equipment because of available visual results. One out of nine A. membranaceus samples was found to be positive of OTA, which was in a good agreement with those obtained from LC–MS/MS analysis. The results demonstrated that the aptamer-based lateral flow assay could be used as a rapid, reliable, cost-effective and robust on-site screening technique for mycotoxins at trace level in complex matrices without special instrumentation.
Keywords: A. membranaceus; Ochratoxin A; Aptamer; Lateral flow strip; On-site detection;

In this work, a sensitive, selective, accurate and precise LC–MS/MS method has been developed for the simultaneous determination of an anti-glaucoma ß-blocker, timolol maleate (TIM) with other co-administered anti-glaucoma drugs of different classes, namely; dorzolamide hydrochloride (DOR), brinzolamide (BRZ) and brimonidine tartrate (BRM) in rabbit aqueous humor (AH) using eslicarbazepine as an internal standard (IS). Liquid-liquid extraction was used for the purification and pre-concentration of analytes from rabbit AH matrix. The chromatographic separation was achieved using a mobile phase consisting of 10 mM ammonium formate pH = 7: methanol: acetonitrile (5: 50: 45, v/v/v) in isocratic mode of elution at a flow rate of 0.8 mL/min on an INERTSIL® C18 ODS-3 column (150 mm × 4.6 mm, 3.5 μm). The method was operated using electrospray ionization source in the positive ionization mode prior to detection by multiple reaction monitoring (MRM) at the following transitions: m/z 317.2 → 261.0 for TIM, m/z 325.1 → 199.0 for DOR, m/z 384.2 → 281.0 for BRZ, m/z 292.1 → 212.0 for BRM and m/z 255.0 → 237.0 for IS. The separation was done in only 3 min and the lower limit of quantitation (LLOQ) was (50 ng/ml) for all cited drugs. A detailed validation of the bio-analytical method was performed as mentioned in US-FDA and EMA guidelines and the standard calibration curves were found to be linear in the range (50–5000 ng/ml) for all drugs with good mean regression coefficient for all drugs.
Keywords: Timolol maleate; Anti-glaucoma drugs; Rabbit aqueous humor; Liquid chromatography; Tandem mass spectroscopy;

A simple, accurate and sensitive ultra fast liquid chromatography coupled with tandem mass spectrometry (UFLC–MS/MS) method was developed for high-throughput determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), fumonisins (FB1 and FB2) and zearalenone (ZEA) in Chinese yam, yam flours and yam-derived products. Mycotoxins were extracted from the samples with methanol-water-formic acid (79:20:1, v/v/v) and no further cleanup step before analysis. After optimization of some crucial parameters including sample preparation, chromatographic separation and MS/MS conditions, the method was successfully validated to exhibit excellent performance in terms of satisfactory linearity (r  ≥ 0.9977), limits of detection (≤0.15 ng mL−1) and quantification (≤0.5 ng mL−1) with good precision (RSD for intra- and inter-day variations of ≤4.65% and 6.31%, respectively), good accuracy (recoveries of 71.0–106.0%) and robustness, together with short run time (8 min/sample). The developed method was applied for simultaneous detection and quantification of the above 8 mycotaxins in 27 batches of Chinese yam and related products collected from different markets and pharmacies in China. The results revealed that 1 normal sample and 4 moldy samples were found to be contaminated with different mycotoxins. The detected concentrations of AFB1 in 2 moldy samples exceeded the regulatory maximum residue levels. The proposed method was capable for simultaneous determination of mycotoxins in this and other types of complex matrices.
Keywords: Multi-mycotoxins; Chinese yam and yam flours; Yam-derived products; High-throughput determination; One-step extraction; UFLC–MS/MS;

Highly sensitive LC–MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study by Raja Reddy Kallem; Bhupathi Jillela; Arun Reddy Ravula; Ramakrishna Samala; Adinarayana Andy; Mullangi Ramesh; JVLN Seshagiri Rao (126-132).
A selective, sensitive and rapid LC–MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200 μL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1 → 105.1 and m/z 375.3 → 201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5 min and the elution of PZ and HZ occurred at 2.53, and 1.92 min, respectively; this was achieved with a mobile phase consisting of 10 mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0 mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51 ng/mL. A linear response function was established for the range of concentrations 0.51–25.2 ng/mL (r  > 0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.
Keywords: Phenelzine; Human plasma; Method validation; LC–MS/MS; Pharmacokinetics;

Pradefovir, a prodrug of PMEA, is under phase 2 clinical trial in China to evaluate its pharmacokinetic and pharmacodynamics after multiple-dose study, with adefovir dipivoxil and tenofovir disoproxil fumarate as positive control. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of pradefovir, PMEA and tenofovir in HBV patient serum. Serum samples were pretreated via simple protein precipitation with methanol and entecavir was used as internal standard. Chromatographic separation was carried out on a Synergi® fusion-RP column (150 mm × 4.6 mm) by gradient elution with methanol and 0.1% formic acid in water (v/v) at a flow rate of 1 mL/min. The analytes were detected in multiple reaction monitoring mode with positive ion electrospray ionization at m/z 424.1/151.0, 274.1/162.2, 288.1/176.1, and 278.1/152.2for pradefovir, PMEA, tenofovir and IS, respectively. The assays were validated according to current bioanalytical guidelines including specificity, linearity (2.0–500 ng/mL for pradefovir and PMEA, 4.0–1000 ng/mL for tenofovir), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to the pharmacokinetic study of pradefovir, adefovir dipivoxil and tenofovir disoproxil fumarate in a set of HBV patients.
Keywords: Pradefovir; PMEA; Tenofovir; LC–MS/MS; Validation;

A miniature extraction envelope containing multiwall carbon nanotubes, diatomite, neutral alumina, and anhydrous magnesium sulfate was developed for the solvent extraction of organophosphorus pesticides in dried ginseng. After the powder of dried ginseng sample was introduced into the envelope, the envelope was heat sealed and sonicated in acetonitrile for the extraction and clean-up of analytes. The resulting extracts were analyzed by an ultrahigh-performance liquid chromatograph with an electrospray ion source and a quadrupole orbitrap mass spectrometer (UHPLC-ESI/Q-Orbitrap MS) under target-MS2 mode, and the analytes were quantified by matrix-match calibration. Finally, the simultaneous identification and quantification of 19 pesticide residues were carried out by the present method. It is noteworthy that in this method the processes of extraction and clean-up for the analytes could be carried out and accomplished in one step. The recoveries and relative standard deviations (RSDs) of analytes were in the range of 82.6–110.8% and 1.0–10.6%, respectively. The limits of detection (LODs) and quantification (LOQs) for analytes were determined to be 0.08–0.29 μg kg−1 and 0.26–0.98 μg kg−1, respectively. Several pesticide residues investigated in this study were found in 4 real samples, with the concentrations lower than the maximum residue limit (0.050 mg kg−1) established by European Union.
Keywords: Carbon nanotube envelope; Organophosphorus pesticides; Dried ginseng root; UHPLC-ESI/Q-Orbitrap MS;

Glycerol and mannitol have the potential to act as plasma volume expanders and have been prohibited as masking agents by the World Anti-Doping Agency (WADA) accordingly. In this study, an improved strategy was developed and validated for the determination of urinary glycerol and mannitol levels simultaneously using a liquid chromatography/tandem mass spectrometry technique within 7 min in an initial testing procedure. For confirmation, mannitol and all possible hexitols (allitol, altritol, galactitol, iditol and sorbitol) that can occur in human urine were baseline separated. This method made use of the derivatization of glycerol and mannitol by benzoyl chloride followed by analysis via LC-ESI-MS/MS with limited sample preparation. The limit of detection (LOD) for glycerol and mannitol was lower than 50 ng/mL. The limit of quantitation (LOQ) for both substances was below 150 ng/mL. The assay was linear from 0.15 to 1000 μg/mL for glycerol and mannitol in human urine. The coefficients of variation of all inter- and intra-assay determinations at three concentration levels (0.5, 500, 900 μg/mL) were better than 13% for glycerol and under 15% for mannitol. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity for both substances.
Keywords: Glycerol; d-mannitol; Quantitation; Derivatization; Doping-control; LC-ESI-MS/MS;

Mercury and selenium binding biomolecules in terrestrial mammals (Cervus elaphus and Sus scrofa) from a mercury exposed area by M.J. Patiño Ropero; N. Rodríguez Fariñas; E. Krupp; R. Mateo; J.J. Berzas Nevado; R.C. Rodríguez Martín-Doimeadios (159-166).
Mercury (Hg) is likely bound to large biomolecules (e.g. proteins) in living organisms, and in order to assess Hg metabolic pathways and possible toxicological effects, it is essential to study these Hg containing biomolecules. However, the exact nature of most metal binding biomolecules is unknown. Such studies are still in their infancy and information on this topic is scarce because the analysis is challenging, mainly due to their lability upon digestion or extraction from the tissue. New analytical methods that allow complex Hg-biomolecules to be analysed intact are needed and only few very recent studies deal with this approach. Therefore, as an initial step towards the characterization of Hg containing biomolecules, an analytical procedure has been optimised using size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) detection. We applied this technique to elucidate the distribution and elution profile of Hg and Se, and some physiological important elements such as Fe, Ni, Zn and Cu, to assess metal binding profiles in liver and kidney samples of red deer (Cervus elaphus) and wild boar (Sus scrofa) who roam freely within the largest Hg mining district on Earth, Almadén in Spain. Elemental fractionation profiles of the extracts from different tissues were obtained using two different SEC columns (BioSep-SEC-S2000 GL 300–1 kDa and Superdex 75 10/300 GL 70–3 kDa). Similar profiles of Hg were observed in red deer and wild boar; however, significant differences were evident for liver and kidney. Moreover, the profiles of Se showed a single peak at high-medium molecular weight in all investigated tissues, while co-elution of Hg with Fe, Ni, Zn and Cu was observed.
Keywords: Mercury; Selenium; Biomolecules; Metallomics; Mammals; Terrestrial animals;

BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid–liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC–MS/MS assay was validated over a concentration range of 0.200–200 ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2 h on ice. The analytes were also stable in human plasma for 25 h at room temperature, 34 days at −20 °C and −70 °C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study.
Keywords: 11-β-Hydroxysteroid-dehydrogenase 1 inhibitor; Metabolite; LC–MS/MS; Assay; Chiral separation; Pharmacokinetics;

Ferromagnetic particles as a rapid and robust sample preparation for the absolute quantification of seven eicosanoids in human plasma by UHPLC–MS/MS by Anna Catharina Suhr; Mathias Bruegel; Barbara Maier; Lesca Miriam Holdt; Alisa Kleinhempel; Daniel Teupser; Stefanie H. Grimm; Michael Vogeser (173-182).
We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to quantitative UHPLC–MS/MS analysis of seven eicosanoids [thromboxane B2 (TXB2), prostaglandin E2 (PGE2), PGD2, 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, arachidonic acid (AA)]. A combination of ferromagnetic particle enhanced deproteination and subsequent on-line solid phase extraction (on-line SPE) realized quick and convenient semi-automated sample preparation—in contrast to widely used manual SPE techniques which are rather laborious and therefore impede the investigation of AA metabolism in larger patient cohorts.Method evaluation was performed according to a protocol based on the EMA guideline for bioanalytical method validation, modified for endogenous compounds. Calibrators were prepared in ethanol. The calibration curves were found to be linear in a range of 0.1–80 ng mL−1 (TXB2, PGE2, PGD2), 0.05–40 ng mL−1 (5-HETE, 11-HETE), 0.5–400 ng mL−1 (12-HETE) and 25–9800 ng mL−1 (AA). Regarding all analytes and all quality controls, the resulting precision data (inter-assay 2.6 %–15.5 %; intra-assay 2.5 %–15.1 %, expressed as variation coefficient) as well as the accuracy results (inter-assay 93.3 %–125 %; intra-assay 91.7 %–114 %) were adequate. Further experiments addressing matrix effect, recovery and robustness, yielded also very satisfying results.As a proof of principle, the newly developed LC–MS/MS assay was employed to determine the capacity of AA metabolite release after whole blood stimulation in healthy blood donors. For this purpose, whole blood specimens of 5 healthy blood donors were analyzed at baseline and after a lipopolysaccharide (LPS) induced blood cell activation. In several baseline samples some eicosanoids levels were below the Lower Limit of Quantification. However, in the stimulated samples all chosen eicosanoids (except PGD2) could be quantified.These results, in context with those obtained in validation, demonstrate the applicability of ferromagnetic particles for the sample preparation for eicosanoids in human plasma. Thus, we conclude that ferromagnetic particle enhanced deproteination is a promising novel tool for sample preparation in LC–MS/MS, which is of particular interest for automation in clinical mass spectrometry, e.g. in order to further address eicosanoid analysis in larger patient cohorts.
Keywords: Magnetic particles; Eicosanoids; Sample preparation; Liquid chromatography; Mass spectrometry; Validation;

An analytical method for determination of the iodine species 3-monoiodotyrosine (MIT), iodide, 3,5-diiodotyrosine (DIT), 3,5-diiodothyronine (3, 5-T2), 3,5,3′-triiodothyronine (T3), and thyroxine (T4) in dog foods and treats is reported. Iodine speciation was carried out using a HPLC method capable of both anion-exchange and reversed-phase retention coupled with inductively coupled plasma mass spectrometry detection (LC-ICP-MS). The method was evaluated by the analysis of the iodine species concentrations in twelve dog foods and treats following enzymatic digestion. The concentrations of MIT, iodide, DIT, T3, and T4 in the samples ranged from 0.64–59.5 μg/g, 0.86–4.05 μg/g, <MDL–74.7 μg/g, <MDL–4.66 μg/g, and <MDL–29.2 μg/g, respectively. The average recoveries based on sample fortification for MIT, iodide, DIT, T2, T3, and T4 were 97%, 114%, 89%, 102%, 90%, and 80%, respectively. An additional analysis was done for DIT, T3, and T4 in the enzyme digest by LC-triple-quadrupole mass spectrometry (LC–MS/MS) to cross-validate the results obtained by LC-ICP-MS. Both methods were in good agreement for the concentrations of DIT, T3, and T4.

High performance curcumin subcritical water extraction from turmeric (Curcuma longa L.) by Mohammad Valizadeh Kiamahalleh; Ghasem Najafpour-Darzi; Mostafa Rahimnejad; Ali Akbar Moghadamnia; Meisam Valizadeh Kiamahalleh (191-198).
Curcumin is a hydrophobic polyphenolic compound derived from turmeric rhizome, which consists about 2–5% of the total rhizome content and is a more valuable component of turmeric. For reducing the drawbacks of conventional extraction (using organic solvents) of curcumin, the water as a clean solvent was used for extracting curcumin. Subcritical water extraction (SWE) experimental setup was fabricated in a laboratory scale and the influences of some parameters (e.g. extraction temperature, particle size, retention time and pressure) on the yield of extraction were investigated. Optimum extraction conditions such as SWE pressure of 10 bar, extractive temperature of 140 °C, particle size of 0.71 mm and retention time of 14 min were defined. The maximum amount of curcumin extracted at the optimum condition was 3.8 wt%. The yield of curcumin extraction was more than 76 wt% with regards to the maximum possible curcumin content of turmeric, as known to be 5%. The scanning electron microscope (SEM) images from the outer surface of turmeric, before and after extraction, clearly demonstrated the effect of each parameter; changes in porosity and hardness of turmeric that is directly related to the amount of extracted curcumin in process optimization of the extraction parameters.
Keywords: Curcumin; Extraction; Subcritical water; Solvent free; Turmeric;

Although many hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) methods have been developed and applied for untargeted metabolite profiling in clinical metabolomics, according to the literature, the suitability of these HILIC-HRMS methods has not been fully evaluated with respect to their performance when they are subjected to statistical analysis. In this study, using a series of human urine samples we investigated the effect of technical variations on multivariate and univariate analysis of the data collected using a previously developed HILIC-HRMS method for untargeted urinary metabolite profiling in clinical metabolomics. The technical variation introduced by sample preparation was more significant than that produced by the HILIC-HRMS method. By using an orthogonal partial least squares (OPLS) model, subtle fold-changes were accurately measured in the urine samples spiked with 13C and 15N isotope labelled amino acids at different concentrations. The robustness of this HILIC method was also evaluated by analysing the obtained data from a single urine sample following manipulation of several primary LC parameters. High reproducibility in the chromatographic performance of three ZIC-pHILIC columns with different batch numbers indicated the reliability of the polymer based zwitterionic stationary phase allowing column replacement without compromising the performance of the method.
Keywords: Clinical metabolomics; hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS); Untargeted metabolite profiling; Technical variation; Isotope labelled amino acids;

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics.The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and β-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods.The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5 °C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.
Keywords: Bevacizumab; Light-scattering; Validation; Aggregation kinetics; Stability;

Sarsasapogenin, a natural compound from Chinese medical herb Anemarrhena asphodeloides Bge., has recently received a great deal of attention due to its various bioactivities. In this study, an easy and applicable liquid chromatography tandem mass spectrometry method for the quantification of sarsasapogenin in rat plasma was developed. Sample preparation was accomplished through a simple one-step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 417.4/273.2 for sarsasapogenin, and 415.2/271.4 for diosgenin (internal standard). The calibration curve was linear over the range of 0.5–500 ng/mL (r = 0.9994), with a lower limit of quantification at 0.5 ng/mL. The RSD of intra- and inter-day precision was below 6.41%, and accuracy ranged from 87.60% to 99.20%. The RSD of matrix effect and recovery yield was within ±15% of nominal concentrations and sarsasapogenin was stable during stability tests. This validated method had been successfully applied to the preclinical pharmacokinetic studies of sarsasapogenin in rats. The half-life (t1/2) was (15.1 ± 2.3), (16.1 ± 3.0) and (15.4 ± 3.9) h after single intragastric administration of 25, 50 and 100 mg/kg sarsasapogenin, respectively. And it was found that, the area under the plasma concentration versus time curve (AUC0-72h) and the maximum plasma concentration (Cmax) were linearly related to dose.
Keywords: Sarsasapogenin; Liquid chromatography tandem mass spectrometry; Rat plasma; Pharmacokinetics;

Simultaneous determination of parecoxib sodium and its active metabolite valdecoxib in rat plasma by UPLC–MS/MS and its application to a pharmacokinetic study after intravenous and intramuscular administration by Meina Liu; Qiuyang Yu; Ping Li; Meng Zhu; Mingming Fang; Bingjun Sun; Mengchi Sun; Yinghua Sun; Peng Zhang; Zhonggui He; Jin Sun; Yongjun Wang; Xiaohong Liu (220-229).
In this study, we developed and validated a new, rapid, specific and sensitive ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to simultaneously determine parecoxib sodium (PX) and its active metabolite, valdecoxib (VX), in rat plasma. Plasma samples were prepared by plasma protein precipitation combined with a liquid-liquid extraction method. The separation was carried out on a Kinetex C18 column (2.1 mm × 50 mm, 2.6 μm) with a gradient elution using methanol (A) and a 2 mM ammonium acetate aqueous solution (B). The analysis was performed in less than 3 min with a flow rate of 0.2 mL/min. Ketoprofen was used as an internal standard (IS). Mass spectrometric detection was conducted with a triple quadrupole detector equipped with electrospray ionization in the negative ion mode (ESI) using multiple reaction monitoring (MRM). The calibration curves were linear over the concentration ranges of 5–4000 ng/mL for PX and 5–2000 ng/mL for VX with all correlation coefficients greater than 0.998. The intra- and inter-day relative standard deviations (RSD) for both analytes were within 15% and the accuracy was within 85–115% at all quality control levels. The mean extraction recoveries for all analytes obtained from three concentrations of QC plasma samples were more than 89.0% efficient. Selectivity, matrix effect, dilution integrity and stability were also validated. The method was successfully used to investigate the pharmacokinetics of PX and VX in rat plasma after intravenous and intramuscular administration of PX.
Keywords: Parecoxib sodium; Valdecoxib; UPLC–MS/MS; Pharmacokinetics; Metabolite; Rat plasma;

Simultaneous analysis method for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin in urine, using C18 poroshell column by Beril Anilanmert; Fatma Çavuş; Ibrahim Narin; Salih Cengiz; Şefika Sertler; Ali Acar Özdemir; Münevver Açikkol (230-241).
Date-rape drugs have the potential to be used in drug-facilitated sexual assault, organ theft and property theft. Since they are colorless, tasteless and odorless, victims can drink without noticing, when added to the beverages. These drugs must be detected in time, before they are cleared up from the biofluids. A simultaneous extraction and determination method in urine for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin (an anticonvulsant and antiepileptic drug) with LC–MS/MS was developed for the first time with analytically acceptable recoveries and validated. A 4 steps liquid-liquid extraction was applied, using only 1.000 mL urine. A new age commercial C18 poroshell column with high column efficiency was used for LC–MS/MS analysis with a fast isocratic elution as 5.5 min. A new MS transition were introduced for barbital. 222.7 > 179.8 with the effect of acetonitrile. Recoveries (%) were between 80.98–99.27 for all analytes, except for GHB which was 71.46. LOD and LOQ values were found in the ranges of 0.59–49.50 and 9.20–80.80 ngmL−1 for all the analytes (except for GHB:3.44 and 6.00 μgmL−1). HorRat values calculated (between 0.25–1.21), revealed that the inter-day and interanalist precisions (RSD% ≤ 14.54%) acceptable. The simultaneous extraction and determination of these 8 analytes in urine is challenging because of the difficulty arising from the different chemical properties of some. Since the procedure can extract drugs from a wide range of polarity and pK a, it increases the window of detection. Group representatives from barbiturates, z-drugs, ketamine, phenytoin and polar acidic drugs (GHB) have been successfully analyzed in this study with low detection limits. The method is important from the point of determining the combined or single use of these drugs in crimes and finding out the reasons of deaths related to these drugs.
Keywords: Drug facilitated sexual assault (DFSA); Determination in urine with LC–MS/MS; GHB; Barbiturate; Zolpidem; Phenytoin;

Direct analysis of [6,6-2H2]glucose and [U-13C6]glucose dry blood spot enrichments by LC–MS/MS by Margarida Coelho; Vera M. Mendes; Inês S. Lima; Fátima O. Martins; Ana B. Fernandes; M. Paula Macedo; John G. Jones; Bruno Manadas (242-248).
A liquid chromatography tandem mass spectrometry (LC–MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-2H2]glucose and [U-13C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-13C6]glucose and an oral glucose load enriched with [6,6-2H2]glucose. The sensitivity is sufficient for analysis of the equivalent to <5 μL of blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments.
Keywords: Glucose kinetics; Isotope dilution measurement; Dried blood spot; Animal model;

Reference ranges for cortisol and α-amylase in mother and newborn saliva samples at different perinatal and postnatal periods by Ana García-Blanco; Máximo Vento; Vicente Diago; Consuelo Cháfer-Pericás (249-255).
This paper describes a reliable analytical method based on Ultra High-Performance Liquid Chromatography coupled to tandem mass spectrometry to determine cortisol in saliva samples from healthy mothers (n = 87) and newborns (n = 65) at different time points: (i) 38 weeks of gestation, (ii) in the immediate postnatal period (48 h) after a term delivery and, (iii) 3 months after delivery. The procedure is characterized by a simple sample treatment employing a sample volume of 25 μL. In addition to this, salivary α-amylase was determined using a commercial kit.We have proposed potential reference ranges in saliva for cortisol (0.7–35 nmol L−1) and α-amylase (2–500 U mL−1) in mothers, and for cortisol (0.1–56 nmol L−1) and α-amylase (0.1–500 U mL−1) in newborn infants. In addition, statistical differences between the two sensitive population groups (mothers and newborns) at the perinatal and postnatal periods were studied. A lower concentration for maternal cortisol was found at 38 weeks of gestation than at 48 h (p = 0.048) or 3 months after delivery (p = 0.021). Similar results were found for the α-amylase determinations. Hence, higher concentrations than could be expected from a chronic stress marker were found at 3 months after delivery than at 38 weeks of gestation (p < 0.001) or 48 h after delivery (p < 0.001).We conclude that this analytical method could be applied to further clinical research on perinatal and postnatal stress, such as threatened preterm labor and/or parenting stress, respectively.
Keywords: Cortisol; α-Amylase; Saliva; Perinatal; Stress;

Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC–MS/MS by Fengjuan Sun; Junjie Ding; Huilan Yu; Runli Gao; Hongmei Wang; Chengxin Pei (256-264).
Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC–MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC–MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC–MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.
Keywords: Human transferrin; Organophosphorus agents; Tyrosine adducts; Serine adducts; Lysine adducts;

Cholinergic components in non-neuronal tissues and cells are proposed as important in maintaining cellular proliferation and immune homeostasis. However, direct quantification of non-neuronal acetylcholine (ACh) in cells has been inefficient. Therefore, we developed a stable method for determination of intracellular ACh.We used ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) with Waters CORTECS chromatographic columns to measure the intracellular ACh in human peripheral blood mononuclear cells (PBMCs). The presence of ACh was validated by RT-qPCR of mRNAs of cholinergic components.This method successfully separated ACh from iso-ACh with highly sensitive precursor/product ion transitions and allowed a faster chromatography run time within 3.5 min. The detection limit of intracellular ACh was 0.005 ng/106 PBMCs. Intra-assay and inter-assay coefficient variations were all <7.0%. Mean recoveries in the samples were between 93.20% and 104.73%. Here, intracellular ACh was stable under multiple storage conditions, partly attributable to mutually stable relationships among cholinergic components. This method was successfully applied in a stroke study and revealed activation of the stroke-induced cholinergic anti-inflammatory pathway.This method allows direct determination of intracellular ACh in various tissues. Evidences of cholinergic activity linking both the nervous system and non-neuronal system can be profiled by assessing ACh.
Keywords: Acetylcholine; Immune cells; Inflammation; Stroke;

Determination of 25-OCH3-PPD and the related substances by UPLC–MS/MS and their cytotoxic activity by Meng Ding; Jingjing Lu; Chen Zhao; Sainan Zhang; Yuqing Zhao (274-280).
20(R)-25-methoxyl-dammarane-3β,12β,20-triol (25-OCH3-PPD) is a promising antitumor compound belonging to triterpenoid saponins isolated from radix notoginseng. A systematic research on the related impurities in raw material of 25-OCH3-PPD has not been conducted. In this study, three impurities obtained by HPLC-ELSD and characterized by 13C NMR and MS were observed in the raw material of 25-OCH3-PPD. Cytotoxic activities of the related substances were also evaluated, of which impurity B with 25-OCH3-PPD showed synergistic inhibitory activity against BGC-823 with IC50 values of 8.33 μM. Furthermore, a rapid and selective UPLC–MS/MS method was developed for simultaneous determination of the principal component and three related substances in the raw material of 25-OCH3-PPD. Multiple reaction monitoring scan mode was used for the quantification of 20(R)-25-OCH3-PPD and its three related substances. The four constituents were separated within 11 min on a BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using a mobile phase comprising methanol and 0.03% formic acid water (82:18, v/v) at a flow rate of 0.2 mL/min. The proposed UPLC–MS/MS method displayed acceptable levels of linearity, precision, repeatability, and accuracy. In addition, the proposed method was successfully applied for the establishment of a rational quality control standard for the raw material of 25-OCH3-PPD.
Keywords: 25-OCH3-PPD; Related impurities; Synergistic anticancer effects; UPLC–MS/MS;

The analysis of veterinary drugs in organic fertilizers is crucial for an assessment of potential risks to soil microbial communities and human health. We develop a robust and sensitive method to quantitatively determine 19 veterinary drugs (amantadine, sulfonamides and fluoroquinolones) in organic fertilizers. The method involved a simple solid-liquid extraction step using the combination of acetonitrile and McIlvaine buffer as extraction solvent, followed by cleanup with a solid-phase extraction cartridge containing polymeric mixed-mode anion-exchange sorbents. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) was used to separate and detect target analytes. We particularly focused on the optimization of sample clean-up step: different diluents and dilution factors were tested. The developed method was validated in terms of linearity, recovery, precision, sensitivity and specificity. The recoveries of all the drugs ranged from 70.9% to 112.7% at three concentration levels, with the intra-day and inter-day relative standard deviation lower than 15.7%. The limits of quantification were between 1.0 and 10.0 μg/kg for all the drugs. Matrix effect was minimized by matrix-matched calibration curves. The analytical method was successfully applied for the survey of veterinary drugs contamination in 20 compost samples. The results indicated that fluoroquinolones had higher incidence rate and mean concentration levels ranging from 31.9 to 308.7 μg/kg compared with other drugs. We expect the method will provide the basis for risk assessment of veterinary drugs in organic fertilizers.
Keywords: Veterinary drugs; Organic fertilizers; UHPLC–MS/MS; Ion-exchange solid-phase extraction;

Quantitation of isocitrate dehydrogenase (IDH)-induced D and L enantiomers of 2-hydroxyglutaric acid in biological fluids by a fully validated liquid tandem mass spectrometry method, suitable for clinical applications by Vianney Poinsignon; Lionel Mercier; Koïchi Nakabayashi; Muriel D. David; Alexandre Lalli; Virginie Penard- Lacronique; Cyril Quivoron; Véronique Saada; Stéphane De Botton; Sophie Broutin; Angelo Paci (290-297).
A recent update of the hallmarks of cancer includes metabolism with deregulating cellular energetics. Activating mutations in isocitrate dehydrogenase (IDH) metabolic enzymes leading to the abnormal accumulation of 2-hydroxyglutaric acid (2-HGA) have been described in hematologic malignancies and solid tumours. The diagnostic value of 2-HGA levels in blood to identify IDH mutations and its prognostic significance have been reported. We developed a liquid chromatography tandem mass spectrometry method allowing a rapid, accurate and precise simultaneous quantification of both L and D enantiomers of 2-HGA in blood samples from acute myeloid leukaemia (AML) patients, suitable for clinical applications. The method was also develop for preclinical applications from cellular and tissues samples. Deuterated (R,S)-2-hydroxyglutaric acid, disodium salt was used as internal standard and added to samples before a solid phase extraction on Phenomenex STRATA™-XL-A (200 mg–3 mL) 33 μm cartridges. A derivatization step with (+)- o,o′-diacetyl-l-tartaric anhydride permitted to separate the two resulting diastereoisomers without chiral stationary phase, on a C18 column combined to a Xevo TQ-MS Waters mass spectrometer with an electrospray ionization (ESI) source. This method allows standard curves to be linear over the range 0.34-135.04 μM with r2 values > 0.999 and low matrix effects (<11.7%). This method, which was validated according to current EMA guidelines, is accurate between-run (<3.1%) and within-run (<7.9%) and precise between-run (<5.3CV%) and within-run (<6.2CV%), and is suitable for clinical and preclinical applications.
Keywords: Biomarker; Isocitrate dehydrogenase mutations; Liquid tandem mass spectrometry; 2-Hydroxyglutaric acid; Endogenous compounds; Oncometabolite;

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of 22 common cephalosporins from the first generation to the fourth generation in pork muscle by liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method. Under the optimized extraction conditions, samples were directly purified through membrane filtration to separate all 22 cephalosporins and the critical pairs of each parent drug were completely separated. Variables affecting the LC–MS/MS were optimized to get a better separation. The excellent selectivity and sensitivity achieved in multiple reactions monitoring (MRM) mode allowed satisfactory confirmation and quantitation for the 22 cephalosporins. The linear range of the 22 cephalosporins is 0.06–100.0 μg/L with good correlation coefficients (r2  > 0.9920). The limits of detection (LODs) and limits of quantitation (LOQs) of these compounds were in the range 0.04–3.0 μg/L and 0.06–10.0 μg/kg, respectively. The average intra-day recoveries at 3 spiked levels (LOQ, 2LOQ, 4LOQ) were all in the range 83.6–113.0% with RSDs (n = 6) lower than 6.5%. The method of LC–MS/MS developed in this study was initially applied to the research of 22 cephalosporins in 12 retail pork samples and proved to be accurate, sensitive, minimum sample pre-treatment, convenient and practical.
Keywords: Liquid chromatography; Tandem mass spectrometry; Cephalosporins; Muscle;