Journal of Pharmaceutical and Biomedical Analysis (v.120, #C)

Integrative drug efficacy assessment of Danggui and European Danggui using NMR-based metabolomics by Zheng-Zheng Zhang; Ma-Li Fan; Xia Hao; Xue-Mei Qin; Zhen-Yu Li (1-9).
Display OmittedDanggui (DG) is a commonly used herbal drug in traditional Chinese medicine, and usually adulterated with European Danggui (EDG) due to the increasing demand. In present study, global metabolic profiling with NMR coupled with integrative drug efficacy evaluation methods was performed to compare and discover underlying blood-enriching regulation mechanisms of DG and EDG on blood deficiency rats induced by acetyl phenylhydrazine (APH). Totally, the contents of 12 key metabolites in serum and 4 in urine of DG group, 7 in serum and 4 in urine of EDG group were significantly reversed in comparison with model group. DG was more effective than EDG as revealed by the relative distance, efficacy index and similarity analysis. The metabolism pathways analysis showed that the better effect of DG maybe related with the regulatory effect on valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies, glycine, serine and threonine metabolism, as well as nicotinate and nicotinamide metabolism. The results presented here showed that metabolomic coupled with efficacy index and similarity analysis made it possible to disclose the subtle biological difference between DG and EDG, which highlight the potential of metabolomic approach to quantitatively compare the pharmacological effect of the herbal drugs.
Keywords: Danggui; European Danggui; Blood enriching effect; Drug efficacy index; Similarity analysis; NMR spectroscopy;

Display OmittedIn this study a selective and high throughput liquid chromatography–mass spectrometry method was developed and validated for the simultaneous quantification of fingolimod (FLD), fampridine (FMP) and prednisone (PDN) in rat plasma using imipramine (IMP) as internal standard (ISTD). In this LC–MS method, following protein precipitation extraction (PPE), the analytes and ISTD were run on XBridge C18 column (150 × 4.6 mm, 5 μm) using gradient mobile phase consisting of 5 mM ammonium formate in water (pH 9.0) and acetonitrile in a flow gradience program. The drug precursor and product ions were monitored on a triple quadrupole instrument that was operated in positive ionization mode. The method was validated over a concentration range of 0.1–100 ng/mL for all the three analytes with relative recoveries ranging from 69 to 82%. The intra and inter batch precision (% CV) across four validation runs were less than 13.4%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) were within ±6.5% of CV values. The method proved to be highly reproducible and sensitive that was successfully applied in a pharmacokinetic study after single dose oral administration to the rats and also in perfusion study sample analysis.
Keywords: Polytherapy; Fingolimod; Fampridine; Phase 4; Multiple sclerosis; Pharmacokinetics; Perfusion;

Display OmittedKinsenoside is a major bioactive constituent isolated from Anoectochilus formosanus and is investigated as an antihyperlipidemic candidate. In this study, a rapid, sensitive, and reliable bioanalytical method was developed for the determination of kinsenoside in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The plasma sample was pretreated with 1% acetic acid, followed by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC silica column (2.1 mm × 100 mm, 3 μm). The mobile phases consisted of 0.1% acetic acid in distilled water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). A gradient program was used at a flow rate of 0.2 mL/min. For mass spectrometric detection, the multiple reaction monitoring mode was used; the MRM transitions were m/z 265.2 →  m/z 102.9 for kinsenoside and m/z 163.3 →  m/z 132.1 for the internal standard (IS) nicotine in the positive ionization mode. A calibration curve was constructed in the range of 2–500 ng/mL. The intra- and interday precision and accuracy were within 5%. The HILIC–MS/MS method was specific, accurate, and reproducible and was successfully applied in a pharmacokinetic study of kinsenoside in rats.
Keywords: Kinsenoside; Antihyperlipidemic agent; LC–MS/MS; Plasma; Pharmacokinetics;

Gene expression of cytokeratin 19 and its molecular detection in human breast cancer cell lines by Umaporn Uawisetwathana; Ekkarat Rodpai; Primchanien Moongkarndi (25-31).
Display Omitted Cytokeratins have been identified as useful tools in oncology diagnostics. In this study, cytokeratin19 (CK19) expression was studied in three human breast cancer cell lines, SKBR3, BT549, and BT474 using RT-PCR. CK19 was expressed in tumor cell of different origin, showing higher expression in invasive breast cancer with ER+ (BT474) than invasive breast cancer with ER (BT549) and breast adenocarcinoma with ER (SKBR3). Two primer sets were used to evaluate CK19 expression. Primer set I (hCK19/1) and primer set II (hCK19/2) were used to amplify the CK19 human gene at a 215 bp and 384 bp, respectively, whereas PBMC and RAW264.7 (mouse macrophage) no detectable PCR products were obtained. The sensitivity for detection was determined by two methods, i.e., cDNA dilution (the dilution of cDNA from RNA of breast cancer cells) and cell dilution (the dilution of breast cancer cells in PBMC). hCK19/2 was more sensitive than hCK19/1. In cDNA dilution, the lower limits of primer set II for detection were 400, 40 and 40 cells for SKBR3, BT549 and BT474 cells, respectively. While in cell dilution all of the 3 breast cancer cells could be detected at 1 cancer cell in 104, 106 and 105 PBMC, respectively. The data supported the possibility that CK19 could be detected and be the marker for breast cancer in patient blood.
Keywords: Cytokeratin19 (CK19); Breast cancer; RT-PCR;

Simultaneous quantification of picfeltarraenins IA and IB in rat plasma by UPLC–MS/MS: Application to a pharmacokinetic study by Xin He; Yingjie Zhang; Hang Gao; Keyan Li; Yazhuo Zhang; Limin Sun; Guizhou Tao (32-37).
Display OmittedA simple and rapid quantitative UPLC–MS/MS method for simultaneous determination of picfeltarraenins IA and IB in rat plasma was developed and validated in accordance with the US FDA Bioanalytical Guidance (2001). Analytes were extracted from rat plasma by using methanol and separated on Agilent ZORBAX SB-C18 (50 mm × 2.1 mm, 1.8 μm) column by using a mobile phase composed of methanol and water (70:30, v/v). Eluents were monitored by ESI tandem mass spectrometry detection with SRM mode using ion transitions m/z 785.4 → 639.5, m/z 815.5 → 669.5, and m/z 763.5 → 455.3 for picfeltarraenin IA, picfeltarraenin IB, and internal standard, respectively. The method was validated over the linear range of 11.5–1150 ng/mL and 13.0–1300 ng/mL. The developed analytical method was applied to support a pharmacokinetic study on simultaneous estimation of picfeltarraenins IA and IB in rats.
Keywords: Picfeltarraenins; Triterpenoid saponins; UPLC–MS/MS; Pharmacokinetic study;

Metabolic profile of naringenin in the stomach and colon using liquid chromatography/electrospray ionization linear ion trap quadrupole-Orbitrap-mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) and LC-ESI-MS/MS by Naiara Orrego-Lagarón; Anna Vallverdú-Queralt; Miriam Martínez-Huélamo; Rosa M. Lamuela-Raventos; Elvira Escribano-Ferrer (38-45).
Display OmittedSeveral biological activities (antioxidant, anti-inflammatory, anticarcinogenic) are attributed to naringenin (NAR)—a predominant flavonoid of citrus fruit and tomato—despite its low bioavailability after ingestion. NAR undergoes extensive metabolism when crossing the gastrointestinal tract, resulting in enteric, hepatic and microbial metabolites, some of them with recognized beneficial effects on human health. This study sought to provide new insights into the metabolism of NAR in regions of the gastrointestinal tract where it has been less studied: the stomach and colon. With this purpose, liquid chromatography coupled with an electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry technique (LC-ESI-LTQ-Orbitrap-MS) was used for an accurate identification of NAR metabolites, and liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a triple quadrupole was used for their identification and quantification. The combination of both analytical techniques provided a broader metabolic profile of NAR. As far as we know, this is the first in-depth metabolic profiling study of NAR in the stomach of mice. Three of the metabolites determined using the LC-LTQ-Orbitrap could not be identified by LC-ESI-MS/MS in stomach perfusion samples: apigenin, 3-(4-hydroxyphenyl) propionic acid and phloroglucinol. The number of colonic metabolites determined using the LTQ-Orbitrap-MS was more than twice the number identified by LC-ESI-MS/MS.
Keywords: Naringenin; Metabolism; Intestinal perfusion; Mice; LC-ESI-LTQ-Orbitrap-MS; LC-ESI-MS/MS;

Optimization of microchip-based electrophoresis for monoclonal antibody product quality analysis revealed needs for extra surfactants during denaturation by Hui Cai; Yuanli Song; Jian Zhang; Ting Shi; Ya Fu; Rong Li; Nesredin Mussa; Zheng Jian Li (46-56).
Display OmittedMicrochip-based electrophoresis has gained increasing popularity in biopharmaceutical development and testing laboratories because of its automation and rapid analysis capabilities. One application of microchip-based electrophoresis is the assessment of size-based variants for product purity analysis. However, monoclonal antibodies analyzed by this technique sometimes exhibited different electrophoretic behaviors. In this study, when three IgG1 and five IgG4 were analyzed using microchip-based electrophoresis under reducing conditions, one of the IgG1s, denoted as mAb1, exhibited an atypical profile attributed to its specific heterogeneity resulting in separation of its heavy chain into two main species. During investigation of the atypical profile, several parameters that were critical to optimal resolution were evaluated, and the data pointed toward incomplete denaturation of mAb1 due to lack of sufficient surfactant in the vendor provided sample buffer (0.7% surfactant). Denaturation studies demonstrated that, although typical antibody profiles could be achieved at 0.7% surfactant for most antibodies analyzed, five out of eight antibodies were not fully denatured until the surfactant concentration reached 0.9% or higher, and mAb1 required a surfactant concentration of 1.3% for complete denaturation. Molecular modeling analysis revealed features in surface charge, hydrophobicity, and structure from mAb1 that led to its unique surfactant concentration-dependent electrophoretic behaviors observed. The optimized method was further evaluated for specificity, linearity, precision, and limit of quantitation for mAb1, and compared with that of conventional CE-SDS.
Keywords: Microchip-based electrophoresis; Monoclonal antibody; Surfactant; Denaturation;

Pharmacokinetic and tissue distribution studies of 1,9-pyrazoloanthrone, a c-Jun-N-terminal kinase inhibitor in Wistar rats by a simple and sensitive HPLC method by Nilesh Sudhakar Ambhore; Karthik Yamjala; Shubhashri Mohire; Kalidhindi Rama Satyanarayana Raju; Shashank Mulukutla; Vishakantha Murthy; Mahesh Tondhawada; Kannan Elango (57-64).
Display OmittedJNK pathway activates c-Jun(s) which are responsible for cell apoptosis; as a result, inhibitors of JNK pathway have the potential to prevent dopaminergic neurons from death and decrease the loss of dopamine in substantia nigra pars compacta (SNpc). Recent in-vitro studies show that 1,9-pyrazoloanthrone (1,9-P) a potent JNK-3 inhibitor prevents the apoptosis of dopaminergic cells of brain. In the present study we formulated liposomes to increase the bioavailability of 1,9-P in the brain and developed a simple, sensitive and selective high performance liquid chromatographic method and validated for the estimation of 1,9-P in Wistar rat plasma and tissue samples. Plasma and tissue samples were extracted by protein precipitation technique using acetonitrile (ACN) and rasagiline as the internal standards. Chromatography was performed on Hibar C18 column with mobile phase of ammonium acetate (10 mM, pH 8.0 adjusted with ammonia) and ACN at a flow rate of 1 mL/min. The lower limit of quantification of the developed method was found to be 2.0 ng/mL and 4.0 ng/g in plasma and tissue samples respectively. The liposomes of 1,9-P administered to animals at the dose equivalent to 15 mg/kg orally demonstrated remarkable absorption into the systemic circulation with maximum concentration (∼7500 ng/mL) within 2.0 h. The order of the area under curve was found to be kidney > liver > brain > lungs > spleen > heart. The liposomes of 1,9-P were rapidly taken up into brain and showed a good brain concentration after 2.0 h; sustenance up to 4.0 h was achieved which is better than 1,9-P solution.
Keywords: 1,9-Pyrazoloanthrone; HPLC method; Pharmacokinetics; Tissue distribution; Validation;

The novel acid degradation products of losartan: Isolation and characterization using Q-TOF, 2D-NMR and FTIR by Avadhesh Kumar Pandey; Ravi Rapolu; Ch. Krishnam Raju; Gururaj Sasalamari; Sanath Kumar Goud; Atul Awasthi; Sameer G. Navalgund; Koduru V. Surendranath (65-71).
Display OmittedForced degradation of losartan potassium in acidic condition resulted into three potential unknown impurities. These unknown degradation products marked as LD-I, LD-II and LD-III were analyzed using a new reverse-phase high performance liquid chromatography (HPLC), eluting at 3.63, 3.73 and 3.91 relative retention times with respect to losartan potassium (LOS) peak. All three were isolated from reaction mass using preparative HPLC and their structures were elucidated using LC–MS/MS, multidimensional NMR and FTIR spectroscopic techniques, as 52,112-dibutyl-54,114-dichloro-11H,51H,71H,111H-1(5,1),7(1,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane,(Z)-52,112-dibutyl-54,114-dichloro-11H,51H,72H,111H-1(5,1),7(2,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane, and 52,112-dibutyl-54,114-dichloro-12H,51H,72H,111H-1(5,2),7(2,5)-ditetrazola-5,11(1,5)-diimidazola-2,8(1,2),3,9(1,4)-tetrabenzenacyclododecaphane, respectively. To best of our knowledge, all three degradation products are novel impurities which are not discussed at any form of publication yet.
Keywords: Losartan potassium; Acidic degradation; LC-QTOF-MS; NMR; FTIR; Novel impurities;

Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the separation of spirostanol saponins by Ling-ling Zhu; Yang Zhao; Yong-wei Xu; Qing-long Sun; Xin-guang Sun; Li-ping Kang; Ren-yi Yan; Jie Zhang; Chao Liu; Bai-ping Ma (72-78).
Display OmittedSpirostanol saponins are important active components of some herb medicines, and their isolation and purification are crucial for the research and development of traditional Chinese medicines. We aimed to compare the separation of spirostanol saponins by ultra-high performance supercritical fluid chromatography (UHPSFC) and ultra-high performance liquid chromatography (UHPLC). Four groups of spirostanol saponins were separated respectively by UHPSFC and UHPLC. After optimization, UHPSFC was performed with a HSS C18 SB column or a Diol column and with methanol as the co-solvent. A BEH C18 column and mobile phase containing water (with 0.1% formic acid) and acetonitrile were used in UHPLC. We found that UHPSFC could be performed automatically and quickly. It is effective in separating the spirostanol saponins which share the same aglycone and vary in sugar chains, and is very sensitive to the number and the position of hydroxyl groups in aglycones. However, the resolution of spirostanol saponins with different aglycones and the same sugar moiety by UHPSFC was not ideal and could be resolved by UHPLC instead. UHPLC is good at differentiating the variation in aglycones, and is influenced by double bonds in aglycones. Therefore, UHPLC and UHPSFC are complementary in separating spirostanol saponins. Considering the naturally produced spirostanol saponins in herb medicines are different both in aglycones and in sugar chains, a better separation can be achieved by combination of UHPLC and UHPSFC. UHPSFC is a powerful technique for improving the resolution when UHPLC cannot resolve a mixture of spirostanol saponins and vice versa.
Keywords: Spirostanol saponins; Ultra-high performance supercritical fluid chromatography; Ultra-high performance liquid chromatography; Chromatographic behavior; Natural products;

Display OmittedA sensitive and rapid analytical method has been validated for the enantiomeric purity determination of l-pidolic acid, a biological lactam and metabolite of glutamic acid commonly found in urine, skin, bones, brain and is available commercially as a food supplement. An efficient, two-step achiral derivatization was implemented which consisted of an alkylation step (using HCl-IPA) followed by an acylation step (using TFAA) of the carboxy and amide functional groups. This allowed detection with high sensitivity using gas chromatography with flame ionization detection. The described procedure employs a CP-Chiralsil-L Val column (25 m × 0.25 mm) at a constant flow rate of 1.5 mL min−1, a gradient temperature program from 80 °C to 160 °C and an injector and detector temperature of 250 °C. The proposed method was validated according to ICH Q2 standards and included such parameters as specificity, system precision, analyst repeatability, intermediate precision, accuracy, linearity, LOD/LOQ and solution stability.
Keywords: Pidolic acid; Enantiomeric purity; Enantioselective gas chromatography; Validation; Derivatization; Amino acids;

Development of a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for nitroimidazoles in edible animal tissues and feeds by Wei Han; Yuanhu Pan; Yulian Wang; Dongmei Chen; Zhenli Liu; Qi Zhou; Liang Feng; Dapeng Peng; Zonghui Yuan (84-91).
Display OmittedThe misuse of nitroimidazoles (NDZs) can lead to NDZs residues in edible animal tissues, which would be harmful to consumer health. To quickly monitor NDZs residues in edible animal tissues and feed, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with a simple sample preparation method and clean-up was developed in the present study. At first, a broad-specificity monoclonal antibody, 1D5, against NDZs has been produced, which the IC50 values of the NDZs, dimetridazole, ipronidazole, ronidazole hydroxydimetridazole, and hydroxyipronidazole, were 4.79 μg L−1, 0.47 μg L−1, 5.97 μg L−1, 23.48 μg L−1, and 15.03 μg L−1, respectively. The limit of detection of the method for the NDZ matrix calibration ranged from 4.2 μg kg−1 to 50.3 μg kg−1 in the feed matrices and from 0.11 μg kg−1 to 4.11 μg kg−1 in the edible animal tissues matrices. The recoveries of the NDZs were in the range of 75.5–111.8%. The CVs were less than 14.4%. A good correlation (r  = 0.9905) between the ELISA and HPLC–MS results of the tissues demonstrated the reliability of the developed ic-ELISA, which makes it a useful tool for screening of NDZs in animal edible tissue and feed.
Keywords: Nitroimidazoles; Monoclonal antibody; Indirect competitive ELISA; Feed; Edible animal tissue;

Display OmittedOpium addiction is one of the main health problems in developing countries and induces serious defects on the human body. In this work, the concentrations of 32 minerals including alkaline, heavy and toxic metals have been determined in the iliac crest bone tissue of 22 opium addicted individuals using inductively coupled plasma-optical emission spectroscopy (ICP-OES). The bone tissues of 30 humans with no physiological and metabolomic diseases were used as the control group. For subsequent analyses, the linear and quadratic discriminant analysis techniques have been used for classification of the data into “addicted” and “non-addicted” groups. Moreover, the counter-propagation artificial neural network (CPANN) has been used for clustering of the data. The results revealed that the CPANN is a robust model and thoroughly classifies the data. The area under the curve for the receiver operating characteristic curve for this model was more than 0.91. Investigation of the results revealed that the opium consumption causes a deficiency in the level of Calcium, Phosphate, Potassium and Sodium in iliac crest bone tissue. Moreover, this type of addiction induces an increment in the level of toxic and heavy metals such as Co, Cr, Mo and Ni in iliac crest tissue. The correlation analysis revealed that there were no significant dependencies between the age of the samples and the mineral content of their iliac crest, in this study. The results of this work suggest that the opium addicted individuals need thorough and restricted dietary and medical care programs after recovery phases, in order to have healthy bones.
Keywords: Iliac crest; Discriminant analysis; Chemometrics; Inductively coupled plasma; Opium;

Determination and quantitation of sildenafil and its major metabolite in the breast milk of a lactating woman by Uwe Wollein; Bernd Schech; Jochen Hardt; Nicholas Schramek (100-105).
Display OmittedA heavily pregnant woman was treated with Revatio® (Sildenafil) against idiopathic pulmonary arterial hypertension, prior to and after her accouchement. To investigate the transfer of sildenafil into breast milk and its metabolism shortly before breastfeeding to the neonatal, a new analytical method was developed and validated, using liquid chromatography tandem mass spectrometry. Additionally, while using linear ion trap scan mode experiments, further metabolites could be identified. Sample preparation was carried out, using solid-phase extraction. For quantification of sildenafil and its major metabolite N-desmethylsildenafil, sildenafil-d8 was used as an internal standard. Within a time frame of 17 h covering two Revatio® intakes and three breast milk samplings, a concentration range from 1.64 to 4.49 ng/ml (sildenafil) and from 1.18 to 1.82 ng/ml (N-desmethylsildenafil) could be observed. The current method proved to be accurate and precise in a very low concentration range and establishes the first reported determination of sildenafil and N-desmethylsildenafil in human breast milk.
Keywords: Sildenafil; Human breast milk; Pulmonary arterial hypertension; Metabolite screening; SPE sample work up;

A critical evaluation of Amicon Ultra centrifugal filters for separating proteins, drugs and nanoparticles in biosamples by Elin Johnsen; Ole Kristian Brandtzaeg; Tore Vehus; Hanne Roberg-Larsen; Vanya Bogoeva; Ornela Ademi; Jon Hildahl; Elsa Lundanes; Steven Ray Wilson (106-111).
Display OmittedAmicon® Ultra centrifugal filters were critically evaluated for various sample preparations, namely (a) proteome fractionation, (b) sample cleanup prior to liquid chromatography mass spectrometry (LC–MS) measurement of small molecules in cell lysate, and (c) separating drug-loaded nanoparticles and released drugs for accurate release profiling in biological samples. (a) Filters of supposedly differing molar mass (M M) selectivity (10, 30, 50 and 100K) were combined to attempt fractionation of samples of various complexity and concentration. However, the products had surprisingly similar M M retentate/filtrate profiles, and the filters were unsuited for proteome fractionation. (b) Centrifugal filtration was the only clean-up procedure in a FDA-guideline validated LC–MS method for determining anti-tuberculosis agents rifampicin and thioridazine in macrophage cell lysate. An additional organic solvent washing step (drug/protein-binding disruption) was required for satisfactory recovery. (c) The centrifugation filters are well suited for separating drugs and nanoparticles in simple aqueous solutions, but significantly less so for biological samples, as common drug–protein binding disruptors can dissolve NPs or be incompatible with LC–MS instrumentation.
Keywords: Centrifugal filters; Proteins; Fractionation; Rifampicin; Thioridazine; Nanoparticles;

The effect of the volume fraction of acetonitrile (φ ACN; 10−2  vol.%) on a retention factors log k of resveratrol and polydatin on different stationary phases.Display OmittedA new high-performance liquid chromatography method using fused-core column for fast separation of resveratrol and polydatin has been developed and used for quality control of nutraceuticals with resveratrol and polydatin content. Retention characteristics (log k) were studied under different conditions on C-18, RP-Amide C-18, Phenyl–hexyl, Pentafluorophenyl (F5) and Cyano stationary phases for both compounds. The effect of the volume fraction of acetonitrile on a retention factors log k of resveratrol and polydatin were evaluated. The optimal separation conditions for resveratrol, polydatin and internal standard p-nitrophenol were found on the fused-core column Ascentis Express ES-Cyano (100 × 3.0 mm), particle size 2.7 μm, with mobile phase acetonitrile/water solution with 0.5% acetic acid pH 3 (20:80, v/v) at a flow rate of 1.0 mL/min and at 60 °C. The detection wavelength was set at 305 nm. Under the optimal chromatographic conditions, good linearity with regression coefficients in the range (r  = 0.9992–0.9998; n  = 10) for both compounds was achieved. Commercial samples of nutraceuticals were extracted with methanol using ultrasound bath for 15 min. A 5 μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery was in the range 83.2–107.3% for both nutraceuticals. The intraday method precision was found satisfactory and relative standard deviations of sample analysis were in the range 0.8–4.7%. The developed method has shown high sample throughput during sample preparation process, modern separation approach, and short time (3 min) of analysis. The results of study showed that the declared content of resveratrol and polydatin varied widely in different nutraceuticals according the producers (71.50–115.00% of declared content).
Keywords: Fused-core columns; HPLC; Chromatography; Nutraceuticals; Resveratrol; Polydatin; Quality control;

Enantioselective analysis of etodolac in human plasma by LC–MS/MS: Application to clinical pharmacokinetics by Carolina de Miranda Silva; Adriana Rocha; Eduardo Tozatto; Lucienir Maria da Silva; Eduardo Antônio Donadi; Vera Lucia Lanchote (120-126).
Display OmittedEtodolac is a non-steroidal anti-inflammatory drug with preferential inhibition of cyclooxigenase-2 and is widely used in the management of pain in patients with inflammatory arthritis. Etodolac is available as a racemic mixture of (−)-(R)-Etodolac and (+)-(S)-Etodolac; cyclooxigenases inhibition is attributed to (+)-(S)-Etodolac. According to our knowledge, this is the first method for determination of etodolac enantiomers in plasma using LC–MS/MS. Plasma extraction were performed with 25 μL of plasma and 1 mL of n-hexane:ethyl acetate (95:5); racemic ibuprofen was used as internal standard. Resolution of enantiomers were performed in a Chiralcel®OD-H column; deprotonated [M-H] and their respective ion products were monitored at transitions of 286 > 242 for etodolac enantiomers and 205 > 161 for ibuprofen. The quantitation limit was 3.2 ng/mL for both enantiomers in plasma. The method was applied to study the pharmacokinetics of etodolac enantiomers after the administration of a 300 and 400 mg dose of racemic drug to a healthy volunteer. Analysis of plasma samples showed higher plasma concentration of (−)-(R)-Etodolacfor both doses (300 mg dose: AUC0–∞49.80 versus 4.55 ug h/mL;400 mg dose: AUC0–∞ 63.90 versus 6.00 ug h/mL) with an (R)-(+)/(S)-(−) ratio of approximately 11.
Keywords: Etodolac; Enantiomers; Pharmacokinetics; LC–MS/MS; Plasma; Healthy volunteer;

Serum metabolomics study of Traditional Chinese medicine formula intervention to polycystic ovary syndrome by Caixia Lu; Xinjie Zhao; Yan Li; Yanjie Li; Chengkun Yuan; Fang Xu; Xiaoyu Meng; Lihui Hou; Guowang Xu (127-133).
Display OmittedPolycystic ovary syndrome (PCOS) is a most common, heterogeneous, complex endocrinopathy disease. Traditional Chinese medicine (TCM) has been used in the treatment of PCOS for many years. However, the mechanism underlying TCM remains obscure and challenging. In this study, 30 PCOS subjects were separated into normoinsulinemic group (NI = 13) and hyperinsulinemic group (HI = 17), and treated for three menstrual cycles with TCM Formula, Bushen Huatan Formula (BHF). A metabolomics approach based on ultra-high-performance liquid chromatography (UPLC) coupled with linear ion trap Orbi-trap mass spectrometer (LTQ Orbi-trap MS) is used to investigate serum metabolic changes of TCM intervention to PCOS. After BHF intervention for three menstrual cycles, the serum levels of glycerophosphorylethanolamine (GPEA), creatine, creatinine decreased in both NI and HI groups. Furthermore, in NI group, the main manifestation was the changes of phospholipid metabolism. While in HI group, lysine, phenol sulfate, phe–phe etc. decreased, and ornithine, proline, betaine, acetylcholine etc. increased. Combined with clinical biochemical data, BHF was proved effective to PCOS by reducing the inflammatory reaction and oxidative stress. This study also illustrates that the LC–MS based metabolomic approach is a helpful tool to evaluate curative effect and to understand the mechanisms of TCM.
Keywords: Metabolomics; LC–MS; Polycystic ovary syndrome; Traditional Chinese medicine; Insulin resistance;

Display OmittedIn this work, an ionic liquid-based ultrasonic-assisted extraction (ILUAE) method was developed to extract caffeoylquinic acids (CQAs) from Flos Lonicerae Japonicae (FLJ). ILUAE parameters were optimized by response surface methodology, including IL concentration, ultrasonic time, and liquid–solid ratio. Optimized ILUAE approach gained the highest extraction yields of 28.53, 18.21, 3.84 mg/g for 3-O-caffeoylquinic acid (C1), 3,5-di-O-caffeoylquinic acid (C2), 3,4-di-O-caffeoylquinic acid (C3), respectively. C1–C3 are the three most abundant CQAs compounds in FLJ. The method showed comparable extraction yield and shorter extraction time compared with conventional extraction techniques. Subsequently, an aqueous two-phase system (ATPS) was applied in extraction solutions. Two trace CQAs, 5-O-caffeoylquinic acid (C4) and 4,5-di-O-caffeoylquinic acid (C5), were significantly enriched with signal to noise values increasing from less than 10 to higher than 1475. The results indicated that ILUAE and ATPS are efficient and environmentally-friendly sample extraction and enrichment techniques for CQAs from herbal medicines.
Keywords: Ionic liquid-based ultrasonic-assisted extraction; Aqueous two-phase system; Response surface methodology; Caffeoylquinic acids; Flos Lonicerae Japonicae;

Display OmittedA simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for simultaneous quantification of naloxone, buprenorphine and its metabolite norbuprenorphine in human plasma. Human plasma samples were extracted using a single step liquid–liquid extraction, and then separated on an Imtakt Unison UK-C18 column (2.1 × 50 mm, 3 μm) using alkaline mobile phases with gradient elution. All of the analytes were detected in positive ion mode using multiple reaction monitoring (MRM). The method was validated and the specificity, linearity, lower limit of quantitation, precision, accuracy, recoveries and stability were determined. The linear range was 20–10000 pg/mL for buprenorphine and norbuprenorphine; and 1–500 pg/mL for naloxone. The correlation coefficient (R 2) values for all three analytes were ≥0.995. The precision and accuracy for intra-day and inter-day were <11.0%. The recoveries were >63% and matrix effects were tracked by the deuterated internal standards (IS) with the IS-normalized matrix factor ranging from 0.96 to 1.33 for all three analytes. The validated method was successfully applied in a clinical pharmacokinetic study with low dose administration of sublingual buprenorphine and naloxone.
Keywords: Buprenorphine; Norbuprenorphine; Naloxone; LC–MS/MS; Human plasma;

Screening HIV-1 fusion inhibitors based on capillary electrophoresis head-end microreactor targeting to the core structure of gp41 by Lihong Liu; Xiaoying Xu; Yanhui Liu; Xuanxuan Zhang; Lin Li; Zhimin Jia (153-157).
Display OmittedIn this paper, we design a microreactor based on electrophoretically mediated microanalysis (EMMA) with capillary electrophoresis (CE) for screening HIV-1 inhibitors that bind to the N-terminal heptad repeat (NHR, N36) region. Initially, a test sample plug is loaded into a capillary filled with buffer solution followed by N36 peptide solution, and the two solutions simultaneously mix by diffusion. Then, voltage is applied, and the sample molecules pass through the N36 peptide zone. The active compounds combine with N36, leading to a loss in the peak height of the active compound. More than 100 traditional Chinese medicine extracts (TCME) were screened, and an extract of Pheretima aspergillum (E. Perrier) (L5) was identified as having potent inhibitory activity. The results showed that L5 could significantly inhibit the HIV-1JR-FL pseudotyped virus infection; the 50% effective concentration (EC50) of L5 was approximately 32.1 ± 1.2 μg/mL, and the 50% cytotoxicity concentration (CC50) value of L5 was 146.9 ± 4.4 μg/mL, suggesting that L5 had low in vitro cytotoxicity on U87-CD4-CCR5 cells. The new method is simple and rapid, is free of antibodies, and does not require tedious processes.
Keywords: Microreactor; Gp41; Six-helix bundles; HIV-1 inhibitors; Traditional Chinese medicines;

Display OmittedPotentiometric and spectrophotometric pH-titration of the multiprotic cytostatics bosutinib for dissociation constants determination were compared. Bosutinib treats patients with positive chronic myeloid leukemia. Bosutinib exhibits four protonatable sites in a pH range from 2 to 11, where two pK are well separated (ΔpK  > 3), while the other two are near dissociation constants. In the neutral medium, bosutinib occurs in the slightly water soluble form LH that can be protonated to the soluble cation LH4 3+. The molecule LH can be dissociated to still difficultly soluble anion L. The set of spectra upon pH from 2 to 11 in the 239.3–375.0 nm was divided into two absorption bands: the first one from 239.3 to 290.5 nm and the second from 312.3 to 375.0 nm, which differ in sensitivity of chromophores to a pH change. Estimates of pK of the entire set of spectra were compared with those of both absorption bands. Due to limited solubility of bosutinib the protonation in a mixed aqueous-methanolic medium was studied. In low methanol content of 3–6% three dissociation constants can be reliably determined with SPECFIT/32 and SQUAD(84) and after extrapolation to zero content of methanol they lead to pK c1  = 3.43(12), pK c2  = 4.54(10), pK c3  = 7.56(07) and pK c4  = 11.04(05) at 25 °C and pK c1  = 3.44(06), pK c2  = 5.03(08) pK c3  = 7.33(05) and pK c4  = 10.92(06) at 37 °C. With an increasing content of methanol in solvent the dissociation of bosutinib is suppressed and the percentage of LH3 2+ decreases and LH prevails. From the potentiometric pH-titration at 25 °C the concentration dissociation constants were estimated with ESAB pK c1  = 3.51(02), pK c2  = 4.37(02), pK c3  = 7.97(02) and pK c4  = 11.05(03) and with HYPERQUAD: pK c1  = 3.29(12), pK c2  = 4.24(10), pK c3  = 7.95(07) and pK c4  = 11.29(05).
Keywords: Dissociation constants; Bosutinib; Spectrophotometric titration; Potentiometric Titration; SQUAD(84) SPECFIT/32; ESAB2M; HYPERQUAD; INDICES; PALLAS;

LC–MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study by Taraka Ramarao Nadella; Vidyadhara Suryadevara; Sasidhar Reddyvallam Lankapalli; Venkata Basaveswara Rao Mandava; Deepti Bandarupalli (168-174).
Display OmittedA simple, rapid, specific and precise liquid chromatography—tandem mass spectrophotometric (LC–MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) with acteonitrile: 10 mM ammonium formate buffer (80:20 v/v) as an isocratic mobile phase with a flow rate of 0.5 mL min−1. Quantitation was performed by transition of 264.1 → 151.1 (m/z) for busulfan and 272.1 → 159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2 ng mL−1 with a 100 μL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100 ng mL−1 (r 2  ≥ 0.9986). Chromatographic separation was achieved within 2.0 min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration.
Keywords: LC–MS/MS; Busulfan; Busulfan d8; Validation; Human Plasma;

Display OmittedChanges in endogenous metabolites in the plasma of streptozotocin (STZ)-induced diabetic rats treated with Ge Gen Qin Lian Decoction (GGQLD) were studied. The endogenous compounds in plasma were detected using ultra high performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). Rats were divided into three groups: control, model, and administration (4.95 g crude drug/kg body weight). After the final administration, plasma samples from the three groups were analyzed using metabonomics. The three sample groups could be clearly distinguished. The administration group exhibited a distinct return to the levels of phytosphingosine and dihydrosphingosine of the control group according to the principal component analysis score, and the corresponding biomarkers were defined. Significant changes in endogenous metabolites, such as dihydrosphingosine, phytosphingosine, cholylglycine, and pantothenic acid, were identified in STZ-induced diabetic rats. These biochemical changes are associated with the metabolism of sphingolipids, fats, and acetyl coenzyme-A, which could be useful to further investigate the characteristics of STZ-induced diabetes mellitus and the therapeutic mechanism of action of GGQLD. This metabonomic analysis could provide a useful starting point toelucidate the therapeutic effects and mechanism of action of GGQLD in diabetes mellitus.
Keywords: UHPLC-Q-TOF; Metabonomics; Diabetes; Ge Gen Qin Lian Decoction;

Aberrant purine metabolism in allergic asthma revealed by plasma metabolomics by Meng Yu; Feng-Xia Cui; Hong-Mei Jia; Chao Zhou; Yong Yang; Hong-Wu Zhang; Gang Ding; Zhong-Mei Zou (181-189).
Sixteen differential metabolites involving into the alteration of six metabolic pathways were identified as potential biomarkers associated with the allergic asthma. And uric acid (P12) and inosine (P13) were recognized as the most important biomarkers of asthma. Inosine (P13) and the other five metabolites (dodecanoic acid (P1), myristic acid (P2), phytosphingosine (P3), sphinganine (P4), and taurocholic acid (P15)) were first reported as biomarkers related to asthma.Display OmittedAsthma is a disease characterized by chronic relapsing airways, and its etiology remains incompletely understood. To better understand the metabolic phenotypes of asthma, we investigated a plasma metabolic signature associated with allergic asthma in ovalbumin (OVA)-sensitized mice by using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Sixteen metabolites were characterized as potential pathological biomarkers related to asthma. Among them, 6 (dodecanoic acid (P1), myristic acid (P2), phytosphingosine (P3), sphinganine (P4), inosine (P13) and taurocholic acid (P15)) were first reported to have potential relevance in the pathogenesis of experimental asthma. The identified potential biomarkers were involved in 6 metabolic pathways and achieved the most entire metabolome contributing to the formation of allergic asthma. Purine metabolism was the most prominently influenced in OVA-induced asthma mice according to the metabolic pathway analysis (MetPA), suggesting that significantly changes in inflammatory responses in the pathophysiologic process of asthma. The metabolites of purine metabolism, especially uric acid (P12) and inosine (P13), may denote their potential as targeted biomarkers related to experimental asthma. The decreased plasma uric acid (P12) suggested that inflammation responses of allergic asthma inhibited the activity of xanthine oxidase in purine metabolism, and manifested the severity of asthma exacerbation. The increased level of inosine (P13) suggests that inflammatory cells induce adenosine triphosphate (ATP) breakdown, resulting in excessive expression of adenosine deaminase (ADA) in the formation of allergic asthma. These findings provided a novel perspective on the metabolites signatures related to allergic asthma, which provided us with new insights into the pathogenesis of asthma, and the discovery of targets for clinical diagnosis and treatment.
Keywords: Allergic asthma; Plasma metabolomics; UPLC-Q-TOF/MS; Purine metabolism; Uric acid; Inosine;

Display OmittedGoiter is an important health problem all over the world and iodine deficiency is its most common cause. Perchlorate, thiocyanate and nitrate (called as major NIS inhibitors) are known to competitively inhibit iodide uptake by the thyroid gland and thus, human exposure to major NIS inhibitors is a public health concern. In this study, an ion chromatographic method for the determination of most common NIS inhibitor ions in drinking waters was developed and validated. This is the first study where an analytical method is used for the determination of major NIS inhibitors in drinking water by an ion chromatography system in a single run. Chromatographic separations were achieved with an anion-exchange column and separated ions were identified by a conductivity detector. The method was found to be selective, linear, precise accurate and true for all of interested ions. The limits of the detections (LOD) were estimated at 0.003, 0.004 and 0.025 mg L−1 for perchlorate, thiocyanate and nitrate, respectively. Possible interference ions in drinking waters were examined for the best separation of NIS inhibitors. The excellent method validation data and proficiency test result (Z-score for nitrate: −0.1) of the FAPAS® suggested that the developed method could be applied for determination of NIS inhibitor residues in drinking waters. To evaluate the usefulness of the method, 75 drinking water samples from Antalya/Turkey were analyzed for NIS inhibitors. Perchlorate concentrations in the samples ranged from not detected (less than LOD) to 0.07 ± 0.02 mg L−1 and the range of nitrate concentrations were found to be 3.60 ± 0.01 mg L−1 and 47.42 ± 0.40 mg L−1. No thiocyanate residues were detected in tested drinking water samples.
Keywords: NIS inhibitors; Drinking water; Ion chromatography; Method development;

Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies by Pauline A. van Schouwenburg; Simone Kruithof; Gertjan Wolbink; Diana Wouters; Theo Rispens (198-201).
Display OmittedComparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.
Keywords: Adalimumab; Immunogenicity; Anti-drug antibodies; Human monoclonal antibodies; Anti-idiotype Antibodies;

Forced degradation, LC–UV, MSn and LC–MS–TOF studies on azilsartan: Identification of a known and three new degradation impurities by Dhiraj Kaushik; Jasmeen Kaur; Vaneet Paul Kaur; Balraj Saini; Yogita Bansal; Gulshan Bansal (202-211).
Four degradation products of Azilsartan formed under different ICH prescribed forced degradation conditions, are characterized through LC–UV, MSn and LC–MS–TOF studies.Display OmittedIn the present study, Azilsartan (AZL) was subjected to ICH recommended forced degradation conditions of hydrolysis, oxidation, dry heat and photolysis. The drug degraded to four degradation products (I–IV) under acidic, alkaline and water hydrolysis and photolysis. All the four degradation products were resolved in a single run on a C-18 column (250 mm × 4.6 mm; 5 μ) with isocratic elution using mobile phase composed of ammonium formate (20 mM, pH 3.0), methanol and acetonitrile (40:5:40% v/v), at a flow rate of 0.8 ml   min−1 at ambient temperature. The products were characterized through +ESI–MSn spectra of AZL and LC–MS–TOF studies as 2-ethoxy-3H-benzo-imidazole-4-carboxylic acid (I), 2-hydroxy-3-[2′-(5-oxo-4,5-dihydro-[1,2,4]oxadiazol-4-ylmethyl]-3H-benzoimidazole-4-carboxylic acid (II, deethylated AZL), 3-[2′-(1H-diazirin-3-yl)-biphenyl]-4-ylmethyl]-2-ethoxy-3H-benzoimidazole-4-carboxylic acid (III), and 3-[4′-(2-ethoxy-benzo-imidazol-1-ylmethyl)-biphenyl-2-yl]-4H-[1,2,4]oxadiazol-5-one (IV, decarboxylated AZL). Product I was found to be a known process related impurity whereas the products II–IV were identified as new degradation impurities. The most probable mechanisms for formation of these degradation products were proposed.
Keywords: Azilsartan medoxomil; LC–MS–TOF; Forced degradation; Mass fragmentation; Impurities;

Quantification of piroxicam and 5′-hydroxypiroxicam in human plasma and saliva using liquid chromatography–tandem mass spectrometry following oral administration by Adriana Maria Calvo; Gabriel Mulinari Santos; Thiago José Dionísio; Maria Paula Marques; Daniel Thomas Brozoski; Vera Lúcia Lanchote; Maria Helena Raposo Fernandes; Flávio Augusto Cardoso Faria; Carlos Ferreira Santos (212-220).
Saliva sampling used to quantify piroxicam and 5′-hydroxypiroxicam is a noninvasive and painless method when compared to sequential blood sampling. For that, a rapid, selective and sensitive liquid chromatography–tandem mass spectrometric method for simultaneous determination of piroxicam and 5′-hydroxypiroxicam in saliva and human plasma was developed and validated. Piroxicam and its major metabolite were separated using a LiChroCART 125-4 RP Select-B Sorbent C18 column using a mixture of methanol and 2% phosphoric acid (pH 2.7) (70:30, v/v) for the mobile phase with a flow injection of 1 mL/min. The run time was 4 min. Volunteers had saliva and blood sampled before, 1, 2, 3, 4, 5, 6, 8, 11, 24, 48 and 72 h after taking a 20 mg oral dose of piroxicam. The pharmacokinetic parameters of piroxicam in plasma samples were as follows: AUC0−72 (64819 h ng/mL), predicted clearance (0.2 L/h), distribution volume (14.8 L), elimination half-life (50.7 h) and saliva/plasma concentration ratio (0.003). The estimation of all pharmacokinetic parameters for 5′-hydroxypiroxicam would require collections beyond 72 h; however, it was possible to quantify the mean maximum concentration (133 ng/mL), time to peak concentration (53.6 h), mean AUC0−72 (6213 h ng/mL), predicted clearance (110.3 L/h) and saliva/plasma concentration ratio (0.04). The developed methods proved effective and sensitive for determining the lower quantification limit of piroxicam in plasma (6.1 ng/mL) and saliva (0.15 ng/mL) and of 5′-hydroxypiroxicam in plasma (1.2 ng/mL) and saliva (0.15 ng/mL).
Keywords: LC–MS/MS; Piroxicam; 5′-Hydroxypiroxicam; Plasma; Saliva;

Display OmittedA novel, selective and robust enantiospecific HPLC method was developed for simultaneous determination of amlodipine and atenolol enantiomers. Box–Behnken design was employed to identify the effect of factors (% ethanol, % diethylamine and flow rate) and their interactions on enantioresolution and analysis time. Chromatography was performed using mobile phase comprising acetonitrile, ethanol and DEA (92:8:0.2% v/v/v) delivered at a flow rate of 1.2 mL min−1 on a Lux Cellulose-4 column. The enantiomers were monitored at a wavelength of 240 nm and separation was achieved within 8 min. The method was validated in terms of specificity, linearity, accuracy, precision, limit of detection and quantification. The method was found to be linear (R 2  ≥ 0.991), accurate (99.8–101.4%) and precise (%RSD ≤ 3%). Additionally, fractional factorial design was used to evaluate the robustness of the method and non-significant intervals for mixture related factors were established using contour profiling. Furthermore, the pertinence of this validated method was established by analyzing three different commercially available formulations. The obtained results confirmed that the proposed method can be extended for routine enantiopurity assay of amlodipine and atenolol in pharmaceutical formulations.
Keywords: Amlodipine; Atenolol; Enantioseparation; Chiral switches; Enantiopurity assay; Liquid chromatography;

Display OmittedMB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100 μL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi® fusion-RP column (150 mm × 4.6 mm, 4 μm) by a gradient-elution with a mobile phase consisting of ammonium formate (1 mM) and methanol at a flow rate of 1 mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100–50000 ng/mL for MB07133, 2–1000 ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18 mg/kg i.v.).
Keywords: Prodrug; MB07133; Cytarabine; Arabinofuranosyluracil; HPLC-MS/MS;

An in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of AChE inhibitors from roots of Coptis chinensis Franch by Hengqiang Zhao; Siduo Zhou; Minmin Zhang; Jinhong Feng; Shanshan Wang; Daijie Wang; Yanling Geng; Xiao Wang (235-240).
Display OmittedIn this study, an in vitro acetylcholinesterase (AChE) inhibition assay based on microplate reader combined with ultrafiltration high performance liquid chromatography-electrospray quadrupole time of flight mass (UF-HPLC-ESI-Q-TOF/MS) was developed for the rapid screening and identification of acetylcholinesterase inhibitors (AChEI) from roots of Coptis chinensis Franch. Incubation conditions such as enzyme concentration, incubation time, incubation temperature and co-solvent was optimized so as to get better screening results. Five alkaloids including columbamine, jatrorrhizine, coptisine, palmatine and berberine were found with AChE inhibition activity in the 80% ethanol extract of C. chinensis Franch. The screened compounds were identified by HPLC-DAD-ESI-Q-TOF/MS compared with the reference stands and literatures. The screened results were verified by in vitro AChE inhibition assays, palmatine showed the best AChE inhibitory activities with IC50 values of 36.6 μM among the five compounds. Results of the present study indicated that the combinative method using in vitro AChE inhibition assay and UF-HPLC-ESI-Q-TOF/MS could be widely applied for rapid screening and identification of AChEI from complex TCM extract.
Keywords: Coptis chinensis Franch; In vitro AChE inhibition assay; UF-HPLC-ESI-Q-TOF/MS; Acetylcholinesterase inhibitor;

An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus by Howard Ramirez-Malule; Stefan Junne; Carlos López; Julian Zapata; Alex Sáez; Peter Neubauer; Rigoberto Rios-Estepa (241-247).
Display OmittedClavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate–imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400 mg/L. The detection and quantification limits were 0.01 and 0.02 mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate–imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.
Keywords: Clavulanic acid; Streptomyces clavuligerus; High performance liquid chromatography method; Fermentation processes; Clavulanate–imidazole;

Four process-related potential new impurities in ticagrelor: Identification, isolation, characterization using HPLC, LC/ESI–MSn, NMR and their synthesis by Neeraj Kumar; Subba Rao Devineni; Prasad Reddy Gajjala; Dharmendra Kumar Gupta; Sandesh Bhat; Rajesh Kumar; Shailendra Kumar Dubey; Pramod Kumar (248-260).
Display OmittedFive process-related impurities were detected in the range of 0.08-0.22% in ticagrelor laboratory batches by HPLC and LC–MS methods. These impurities were named as TIC Imp-I, -II, -III, -IV and -V. Four of these impurities, TIC Imp-I to -IV were unknown and have not been reported previously. Based on LC–ESI/MSn study, the chemical structures of new impurities were presumed as (1S,2S,3S,5S)-3-(2-hydroxyethoxy)-5-(7-amino-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d] pyrimidin-3-yl)cyclopentane-1,2-diol (TIC Imp-I), (1S,2S,3S,5S)-3-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylsulfinyl)-3H-[1,2,3]triazolo [4,5-d]pyrimidin-3-yl)-5-(2-hydroxyethoxy)cyclopentane-1,2-diol (TIC Imp-II), (1S,2R,3S,4S)-4-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2,3-triol (TIC Imp-III) and (3S,5S)-3-(7-((1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-5-(2-hydroxyethoxy)cyclopentane-1,2-diol (TIC Imp-IV). The unknown impurities were isolated from enriched crude sample by column chromatography and preparative HPLC. The complete spectral analysis, MS, 1D NMR (1H, 13C and DEPT), 2D NMR (HSQC and HMBC) and IR confirmed the proposed chemical structures of impurities. Identification, isolation, structural characterization, prospects for the formation of impurities and their synthesis were first reported in this paper.
Keywords: Ticagrelor; Impurities; Identification; Isolation; LC–ESI/MS; NMR;

Display OmittedSulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75 min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3 → 97.0) and cortisol (m/z 407.2 → 331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2 pmol DHEA-S and the calibration curve was linear from 0.2 to 200 pmol. The intra-day and inter-day accuracy and precision was <11.7%. DHEA-S in the quality control samples was stable at room temperature, 4 °C, and −20 °C. The cytosolic matrix (20–100 μg cytosolic protein) did not affect DHEA-S quantification. Our UPLC–MS/MS method was applied to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. The optimal levels of MgCl2 and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Reducing agents, including 2-mercaptoethanol and DL-dithiothreitol, did not affect the enzyme activity. A linear relationship existed between DHEA sulfation and amount of human liver cytosol (20–200 μg cytosolic protein) or incubation time (5–30 min). This UPLC–MS/MS approach is safer, easier, and faster than existing radiometric-based sulfotransferase enzyme assays, and it is the first UPLC–MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.
Keywords: Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Human liver cytosol; Sulfotransferase 2A1; Ultra-high performance liquid chromatography–tandem mass spectrometry;

Display OmittedThis paper describes a new, improved systematic qualitative analysis of yucca saponins in commercial products by combined use of high-pressure liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Three groups with four saponins in each were completely resolved by HPLC-ELSD under C18 reversed-phase conditions using a linear gradient composed of methanol and water. The selectivity of the described HPLC-ELSD method is given by the sapogenin differences at C12 (carbonyl group) and at C2 (hydroxyl group), followed by the presence or absence of an exomethylene group at C25, and the composition and length of the oligosaccharidic chain at C3 in the sapogenins. The saponins were identified by chemical and spectroscopic methods including MALDI-TOF MS1 and MS2, high resolution MS, and one and bi-dimensional nuclear magnetic resonance (NMR) experiments. Among the twelve saponins identified, eleven were previously reported, and one is reported for the first time in Yucca schidigera. An identification flowchart procedure based on the analysis of the sodium adducts of intact saponins [M+Na]+ and the oligosaccharidic chain ions obtained in the MALDI-TOF MS1 and the MS2 spectrums, was developed for the analysis of Y. schidigera saponins. Two intact isomeric saponins were differentiated by this method, which could also be applied to the structural assignment of other steroid and triterpenic saponins. Using the four-major saponins as reference standards, a C18 reversed-phase HPLC-ELSD method was validated for their specific analysis in commercial extracts of Y. schidigera. Finally, the applicability of the HPLC-ELSD method for the relative quantification of yucca saponins is discussed.
Keywords: Yucca schidigera; Steroid saponins; MALDI-TOF; HPLC-ELSD; Qualitative analysis; Quantitative analysis;

Simple alternative to sialic acid determination in meningococcal polysaccharides W or Y by M. Morduš; A. Štimac; M. Brgles; T. Kurtović; B. Halassy (283-289).
Display OmittedPhysicochemical methods are the primary tests used to ensure that batches of meningococcal polysaccharide (PS) antigens are manufactured consistently to those shown to be safe and effective in clinical trials. Although modern physicochemical methods of analysis providing structural information about the antigens have been developed and used, simpler assays, which can be readily validated, are still in use for polysaccharide batch release. The simple and cheap method for Neisseria meningitidis serogroup W or Y polysaccharide (MenW or MenY PS) content quantification has been developed. This colorimetric method is based on the galactose or glucose quantification in MenW or MenY PS hydrolysate, respectively. Intra- and inter-assay precision and accuracy of the novel method have been demonstrated, in comparison to the same properties of the current regulatory approved method for the same purpose — sialic acid quantification. We provided the calculation of the possible future regulatory requirement for the galactose or glucose content in MenW or MenY PS, respectively, and revealed in detail the stoichiometric calculation behind it.
Keywords: Neisseria meningitides; Meningococcal vaccine; Serogroup W polysaccharide; Serogroup Y polysaccharide; Polysaccharide content quantification;

Development and validation of HPLC method with fluorometric detection for quantification of bisnaphthalimidopropyldiaminooctane in animal tissues following administration in polymeric nanoparticles by Marcela A. Segundo; Vera L.R.G. Abreu; Marcelo V. Osório; Sonia Nogueira; Paul Kong Thoo Lin; Anabela Cordeiro-da-Silva; Sofia A.C. Lima (290-296).
Display OmittedA simple, sensitive and specific high-performance liquid chromatography method for the quantification of bisnaphthalimidopropyldiaminooctane (BNIPDaoct), a potent anti-Leishmania compound, incorporated into poly(d,l-lactide-co-glycolic acid) (PLGA) nanoparticles was developed and validated toward bioanalysis application. Biological tissue extracts were injected into a reversed-phase monolithic column coupled to a fluorimetric detector (λ exc  = 234 nm, λ em  = 394 nm), using isocratic elution with aqueous buffer (acetic acid/acetate 0.10 M, pH 4.5, 0.010 M octanesulfonic acid) and acetonitrile, 60:40 (v/v) at a flow rate of 1.5 mL min−1. The run time was 6 min, with a BNIPDaoct retention time of 3.3 min.Calibration curves were linear for BNIPDaoct concentrations ranging from 0.002 to 0.100 μM. Matrix effects were observed and calibration curves were performed using the different organ (spleen, liver, kidney, heart and lung) extracts. The method was found to be specific, accurate (97.3–106.8% of nominal values) and precise for intra-day (RSD < 1.9%) and inter-day assays (RSD < 7.2%) in all matrices. Stability studies showed that BNIPDaoct was stable in all matrices after standing for 24 h at room temperature (20 °C) or in the autosampler, and after three freeze–thaw cycles. Mean recoveries of BNIPDaoct spiked in mice organs were >88.4%. The LOD and LOQ for biological matrices were ≤0.8 and ≤1.8 nM, respectively, corresponding to values ≤4 and ≤9 nmol g−1 in mice organs. The method developed was successfully applied to biodistribution assessment following intravenous administration of BNIPDaoct in solution or incorporated in PLGA nanoparticles.
Keywords: Poly(d,l-lactide-co-glycolic acid); Bisnaphthalimido-propyldiamino-octane; Anti-leishmanial compound; Monolithic column; Fluorometry;

Design of experiments for enantiomeric separation in supercritical fluid chromatography by Elodie Landagaray; Claude Vaccher; Saïd Yous; Emmanuelle Lipka (297-305).
Display OmittedA new chiral melatoninergic ligand, potentially successor of Valdoxan®, presenting an improved pharmacological profile with regard to agomelatine, was chosen as a probe for a supercritical fluid chromatographic separation carried-out on an amylose tris[(S)-1-α-methylbenzylcarbamate] based stationary phase.The goal of this work was to optimize simultaneously three factors identified to have a significant influence to obtain the best resolution in the shortest analysis time (i.e., retention time of the second eluting enantiomer) for this chiral compound.For this purpose a central circumscribed composite (CCC) design was developed with three factors: the flow-rate, the pressure outlet and the percentage of ethanol to optimize of two responses: shortest analysis time and best resolution. The optimal conditions obtained via the optimizer mode of the software (using the Nelder-Mead method) i.e., CO2/EtOH 86:14 (v:v), 104 bar, 3.2 mL min−1 at 35 °C lead to a resolution of 3.27 in less than 6 min. These conditions were transposed to a preparative scale where a concentrated methanolic solution of 40 mM was injected with a sample loop of 100 μL. This step allowed to separate an amount of around 65 mg of racemic melatonin ligand in only 3 h with impressive yields (97%) and enantiomeric excess (99.5%).
Keywords: Chiral separation; Experimental design; Melatoninergic ligands; Naphtalens; Optimization; Polysaccharide chiral stationary phase;

Chiral separation of new designer drugs (Cathinones) on chiral ion-exchange type stationary phases by Denise Wolrab; Peter Frühauf; Alena Moulisová; Martin Kuchař; Christopher Gerner; Wolfgang Lindner; Michal Kohout (306-315).
Display OmittedWe present the enantioseparation of new designer drugs from the cathinone family on structurally different chiral ion-exchange type stationary phases. A novel strong cation-exchange type chiral stationary phase was synthesized and its performance compared with previously reported ion-exchange type chiral stationary phases. The influence of structural elements of the chiral selectors on their chromatographic performance was studied and the possibilities of tuning chromatographic parameters by varying the polarity of the employed mobile phases were determined. Evidence is provided that a change in mobile phase composition strongly influences the solvation shell of the polarized and polarizable units of the selectors and analytes, as well as ionizable mobile phase additives. Furthermore, the structural features of the selectors (e.g. the size of aromatic units and their substitution pattern) are shown to play a key role in the effective formation of diastereomeric complexes with analytes. Thus, we have achieved the enantioseparation of all test analytes with a mass spectrometry-compatible mobile phase with a chiral strong cation-exchange type stationary phase.
Keywords: New designer drugs; Cathinones; Enantiomer separation; Chiral stationary phase; Chiral ion exchanger;

Determination of pinostilbene in rat plasma by LC–MS/MS: Application to a pharmacokinetic study by Wan Chen; Samuel Chao Ming Yeo; Xue Fen Chuang; Hai-Shu Lin (316-321).
Display OmittedPinostilbene (3-methoxyresveratrol or trans-3,4′-dihydroxy-5-methoxystilbene) is a naturally occurring monomethylether analogue of resveratrol (trans-3,5,4′-trihydroxystilbene) that exhibits various pharmacological activities. To further examine its medicinal potential, a sensitive LC–MS/MS method was developed and validated for the determination of pinostilbene in rat plasma. Heavy Isotope labelled resveratrol was used as an internal standard. The ESI was operated in its negative ion mode while pinostilbene and resveratrol were measured by multiple reaction monitoring (MRM) using precursor-to-product ion transitions of m/z 241 → 181 and m/z 233 → 191, respectively. This LC–MS/MS method had excellent selectivity, sensitivity (LLOQ = 1 ng/ml), accuracy (both intra- and interday analytical recovery within 100 ± 15%) and precision (both intra- and interday RSD < 15%). The matrix effect was insignificant. The pharmacokinetics of pinostilbene was subsequently profiled in Sprague–Dawley rats. Upon intravenous administration (5 or 10 mg/kg), pinostilbene displayed rapid clearance (Cl = 129 ± 42 or 107 ± 31 ml/min/kg) and extremely short mean transit time (MTT = 6.24 ± 0.41 or 8.52 ± 1.38 min). After oral dosing (50 mg/kg), the bioavailability of pinostilbene was limited but highly erratic (F  = 1.87 ± 2.67%). Pharmacokinetic comparison among pinostilbene, resveratrol and some resveratrol analogues suggested that stilbenes with meta-hydroxyl group(s) may be associated with metabolic instability and subsequently suffer from rapid clearance and low oral bioavailability. The information obtained from this study will facilitate further exploration on pinostilbene as well as other resveratrol analogues.
Keywords: Pinostilbene; Resveratrol; LC–MS/MS; Pharmacokinetics;

Display OmittedA sensitive and specific method was developed and validated for the quantitation of maytansinoid (DM1) in human serum using on-line solid phase extraction (SPE)—liquid chromatography–tandem mass spectrometry (LC–MS/MS). Because DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum, samples were pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). The resulting samples were diluted with acetonitrile prior to the on-line solid phase extraction (SPE) on a C18 cartridge. A C18 (150 × 4.6 mm ID 3 μm particle size) column was used for chromatographic separation with a 10.0 min HPLC gradient and DM1-NEM was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. DM1 concentrations were back-calculated from DM1-NEM amount found in the human serum samples. The quantitation range of the method was 0.200–200 ng/mL when using 0.25 mL serum. Within-run day precisions (n  = 6) were 0.9–4.4% and between-run day (3 days runs; n  = 18) precisions 2.5–5.6%. Method biases were between 3.5–14.5% across the whole calibration range. DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. Finally, the assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload.
Keywords: Maytansinoid; Antibody drug conjugate; LC–MS/MS; On-line SPE and human serum;

Discriminating nicotine and non-nicotine containing e-liquids using infrared spectroscopy by E. Deconinck; J.L. Bothy; S. Barhdadi; P. Courselle (333-341).
Display OmittedIn a few countries, including Belgium, nicotine-containing e-cigarettes and e-liquids are considered medicines, and therefore cannot freely be sold, but should be distributed in a pharmacy. The fact that in the neighbouring countries these products are freely available, poses a problem for custom personnel, the more the nicotine content of the products is not always labelled, especially when they are bought through internet. Therefore there is a need for easy-to-use equipment and methods to perform a first on site screening of intercepted samples, both for border control as to check label compliance of the sample.The use of attenuated total reflectance-infrared spectroscopy (ATR-IR) and near infrared spectroscopy (NIR), combined with chemometrics was evaluated for the discrimination between nicotine containing and non-nicotine containing samples.It could be concluded that both ATR-IR and NIR could be used for the discrimination when combined with the appropriate chemometric techniques. The presented techniques do not need sample preparation and result in models with a minimum of false negative samples. If a large enough training set can be established the interpretation can be fully automated, making the presented approach suitable for on-site screening of e-liquid samples.
Keywords: ATR-IR; Nicotine; e-Cigarettes; Chemometrics; Mobile detection approaches;

An iterative approach for compound detection in an unknown pharmaceutical drug product: Application on Raman microscopy by Mathieu Boiret; Nathalie Gorretta; Yves-Michel Ginot; Jean-Michel Roger (342-351).
Display OmittedRaman chemical imaging provides both spectral and spatial information on a pharmaceutical drug product. Even if the main objective of chemical imaging is to obtain distribution maps of each formulation compound, identification of pure signals in a mixture dataset remains of huge interest. In this work, an iterative approach is proposed to identify the compounds in a pharmaceutical drug product, assuming that the chemical composition of the product is not known by the analyst and that a low dose compound can be present in the studied medicine. The proposed approach uses a spectral library, spectral distances and orthogonal projections to iteratively detect pure compounds of a tablet. Since the proposed method is not based on variance decomposition, it should be well adapted for a drug product which contains a low dose product, interpreted as a compound located in few pixels and with low spectral contributions. The method is tested on a tablet specifically manufactured for this study with one active pharmaceutical ingredient and five excipients. A spectral library, constituted of 24 pure pharmaceutical compounds, is used as a reference spectral database. Pure spectra of active and excipients, including a modification of the crystalline form and a low dose compound, are iteratively detected. Once the pure spectra are identified, multivariate curve resolution-alternating least squares process is performed on the data to provide distribution maps of each compound in the studied sample. Distributions of the two crystalline forms of active and the five excipients were in accordance with the theoretical formulation.
Keywords: Raman microscopy; Orthogonal projection; Spectral angle mapper; Identification; Low dose compound;

Characterization of forced degradation products and in silico toxicity prediction of Sofosbuvir: A novel HCV NS5B polymerase inhibitor by Debasish Swain; Gananadhamu Samanthula; Shweta Bhagat; P.V. Bharatam; Venkatakrishna Akula; Barij N. Sinha (352-363).
Display OmittedSofosbuvir is a direct acting antiviral medication used to treat Hepatitis C viral infection. The present study focuses on the degradation behavior of the drug under various stress conditions (hydrolysis, oxidative, thermal and photolytic) as per International Conference on Harmonization (ICH Q1A (R2)) guidelines. A high performance liquid chromatographic system (HPLC) was used to develop a selective, precise and accurate method for separating all the degradation products. The separation was achieved on a Sunfire™ C18 (150 mm × 4.6 mm × 5 μm) stationary phase with a mobile phase of 10 mM ammonium acetate (pH 5.0) buffer and acetonitrile in gradient elution mode. A quadrupole-time of flight mass analyzer equipped with an electrospray ionization technique was used to propose the structural information based on the MS/MS and accurate mass measurements. Seven degradation products were identified and characterised by LC-ESI-QTOF–MS/MS. In silico toxicity of the drug and its degradation products was determined using TOPKAT and DEREK toxicity prediction softwares. The proposed method was validated as per the ICH Q2 guidelines.
Keywords: Sofosbuvir; Degradation behavior; LC-ESI-QTOF–MS/MS; In silico toxicity;

Display OmittedTongmai Yangxin (TMYX) Pill is a traditional Chinese patent medicine, composed of eleven Chinese medicinal herbs. It has been used to treat coronary heart disease for several decades. In this study, six male Sprague-Dawley rats were dosed orally with TMYX methanol extract, and a serum pharmacochemistry technique was used to screen absorbed bioactive compounds by UPLC/Q-TOF-MS. By comparing MS spectra to the published literature data, 40 bioactive components were identified. The results indicated that almost 45% of the absorbed compounds were from Radix Glycyrrhizae (GC). Subsequently, a reliable HPLC method was used to determine the concentrations of liquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, and glycyrrhetinic acid in rat plasma following oral administration of GC or the combination of GC and Ramulus Cinnamomi (GZ). The results showed that GZ enhanced the absorption of four bioactive components: liquiritigenin, isoliquiritigenin, glycyrrhizic acid, and glycyrrhetinic acid. The data demonstrate that herb combination in TMYX Pill exhibit a synergistic action.
Keywords: Tongmai Yangxin Pill; Serum pharmacochemistry technique; Pharmacokinetics; Combination; Radix Glycyrrhizae; Ramulus Cinnamomi;

Display OmittedBuddlejasaponin IV (BS-IV), a natural triterpene saponin isolated from several herbal plants, has drawn a lot of attention for its anti-inflammatory, antinociceptive, antihyperlipidemia, and antitumor activities. In this study, a simple and sensitive method for determination of BS-IV in rat plasma was developed for the first time, using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Tenacissoside I was used as an internal standard (IS). Separation was achieved on an Agilent Extend-C18 column with gradient elution using methanol–water as mobile phase at a flow rate of 400 μL/min. A triple quadrupole mass spectrometer operating in the positive/negative ion-switching electrospray ionization mode with selection reaction monitoring (SRM) was used to determine BS-IV and IS transitions of 941.4 → 779.5 and 815.5 → 755.5, respectively. The lower limit of quantification was 3.00 ng/mL with a linear range of 3.0–3000 ng/mL. The intra- and inter-day precisions were both ≤10.4% for BS-IV, and the average intra- and inter-day accuracies ranged from −7.2% to 6.7%. The validated assay was successfully applied to a pharmacokinetic study of BS-IV following oral administration of 3, 6, 12 mg/kg and an intravenous administration of 0.9 mg/kg to rats.
Keywords: Buddlejasaponin IV; UHPLC-MS/MS; Rat plasma; Pharmacokinetics;

Separation of indole-3-carbinol and condensation products of indol-3-carbinol CP1, CP2 and CP3 performed on the column Kinetex 5 μ XB-C18 100A.Display OmittedIndole-3-carbinol is a natural glucosinolate known for prevention of human breast, prostate and other types of cancer and it started to be used in commercial preparations, as food supplements. However no analytical method has been proposed for quality control of nutraceuticals with this substance yet. In this paper a new high-performance liquid chromatography (HPLC) method using core–shell column for separation of indole-3-carbinol and its condensation/degradation products was developed and used for the quantitative determination of indole-3-carbinol in nutraceuticals. Separation of indole-3-carbinol, its condensation/degradation products and internal standard ethylparaben was performed on the core–shell column Kinetex 5 μ XB-C18 100A (100 × 4.6 mm), particle size 5.0 μm, with mobile phase acetonitrile/water according to the gradient program at a flow rate of 1.25 mL min−1 and at temperature 50 °C. The detection wavelength was set at 270 nm. Under the optimal chromatographic conditions good linearity of determination was achieved. Available commercial samples of nutraceuticals were extracted with 100% methanol using ultrasound bath. A 5-μL sample volume of the supernatant was directly injected into the HPLC system. The developed method provided rapid and accurate tool for quality control of nutraceuticals based on cruciferous vegetable extracts with indole-3-carbinol content. The presented study showed that the declared content of indole-3-carbinol significantly varied in the different nutraceuticals available on the market. Two analyzed preparations showed the presence of condensation/degradation products of indole-3-carbinol which were not officially declared by the manufacturer. Moreover, further two analyzed nutraceutical preparations showed absolutely no content of declared amount of indole-3-carbinol.
Keywords: Indole-3-carbinol; Nutraceuticals; Condensation products; Liquid chromatography; Core–shell columns; Quality control;

A new modified wetting test and an alternative disintegration test for orally disintegrating tablets by Patrick Hooper; Jason Lasher; Kenneth S. Alexander; Gabriella Baki (391-396).
Display OmittedIndustrial manufacturing of solid oral dosage forms require quality tests, such as friability, hardness, and disintegration. The United States Pharmacopeia (USP) disintegration test uses 900 mL of water. However, recent studies of orally disintegrating tablets (ODTs) have shown that this volume does not accurately portray the oral environment. In our study, various tests were conducted with a more moderate amount of water that accurately resembles the oral environment. A simulated wetting test was performed to calculate the water absorption ratio. Results showed that wetting was comparable to disintegration. Although the wetting test worked for most types of ODTs, it had limitations that produced inaccurate results. This led to the use of a modified shaking water bath test. This test was found to work for all types of ODT products and was not subject to the limitations of the wetting test. The shake test could provide disintegration times rather than water permeation times; however, it could not be used to calculate the water absorption ratio. A strong correlation was observed between the standardized shake test and the USP disintegration times for the tablets. This shake test could be used during the development stages and quality tests for ODTs with relative ease.
Keywords: Wetting test; Disintegration; ODT; Orally disintegrating tablet; Water absorption; Oral disintegration;

New sorbent materials for selective extraction of cocaine and benzoylecgonine from human urine samples by Renata Bujak; Renata Gadzała-Kopciuch; Alicja Nowaczyk; Joanna Raczak-Gutknecht; Marta Kordalewska; Wiktoria Struck-Lewicka; Małgorzata Waszczuk-Jankowska; Ewa Tomczak; Michał Kaliszan; Bogusław Buszewski; Michał J. Markuszewski (397-401).
Display OmittedAn increase in cocaine consumption has been observed in Europe during the last decade. Benzoylecgonine, as a main urinary metabolite of cocaine in human, is so far the most reliable marker of cocaine consumption. Determination of cocaine and its metabolite in complex biological samples as urine or blood, requires efficient and selective sample pretreatment. In this preliminary study, the newly synthesized sorbent materials were proposed for selective extraction of cocaine and benzoylecgonine from urine samples. Application of these sorbent media allowed to determine cocaine and benzoylecgonine in urine samples at the concentration level of 100 ng/ml with good recovery values as 81.7% ± 6.6 and 73.8% ± 4.2, respectively. The newly synthesized materials provided efficient, inexpensive and selective extraction of both cocaine and benzoylecgonine from urine samples, which can consequently lead to an increase of the sensitivity of the current available screening diagnostic tests.
Keywords: New sorbent media; Cocaine; Benzoylecgonine; Human urine; Forensic analysis;

Chiral separation of tedizolid using charge single isomer derivatives of cyclodextrins by capillary electrokinetic chromatography by Katarzyna Michalska; Ewa Gruba; Judyta Cielecka-Piontek; Elżbieta Bednarek (402-412).
Display OmittedA method to enantioseparate tedizolid (TED), the second analogue after linezolid (LIN) in a truly new class of antibacterial agents, the oxazolidinones, was developed based on capillary electrokinetic chromatography using cyclodextrin as chiral pseudophase (CD-cEKC). The single isomer R-tedizolid possesses one chiral centre at C5 of the oxazolidinone ring, which is associated with the antibacterial activity of the drug. Tedizolid enantiomers are non-charged and therefore require the use of charged cyclodextrins (CCDs) as carrier hosts to achieve a velocity difference during migration. During method development, hydrophilic anionic single-isomer and moderately hydrophobic and hydrophobic cyclodextrins were tested, including heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD), oktakis-(2,3-diacetyl-6-sulfo)-γ-cyclodextrin (ODAS-γ-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD). Only CDs that have acetyl groups at the C2 and C3 positions with seven (HDAS-β-CD) or eight (ODAS-γ-CD) residues of glucopyranose units provided baseline separation of the tedizolid enantiomers with the addition of organic solvent. During the experiments, different organic solvents were tested, such as methanol, acetonitrile, tetrahydrofuran, which varied in their abilities to donate or accept protons. The best enantiomer separation results were obtained using the CD-cEKC method with 37.5 mM HDAS-β-CD dissolved in 50 mM formic buffer (pH 4.0) with the addition of acetonitrile (81.4:18.6, v/v) at 27 ºC, normal polarity, and 12 kV. Finally, the apparent binding constants for each enantiomer—HDAS-β-CD pair were calculated. Moreover, in order to evaluate the behaviour of TED and LIN enantiomers relative to chiral selector, enantioselective interactions towards the precursors of TED and LIN isomers were also investigated.
Keywords: Antibacterial agent; CD-cEKC; Cyclodextrin; Enantioseparation; Synthesis; Tedizolid;

Quantitative assay of capreomycin oleate levels in a drug formulation for inhalation with a fully validated HPLC method by Federica Ianni; Aurélie Schoubben; Domenico Montesano; Nathalie Wauthoz; Lina Cossignani; Roccaldo Sardella; Benedetto Natalini (413-418).
Display OmittedCapreomycin sulfate (CS), a mixture of 4 closely related compounds (powder mainly comprised of 2 forms), commonly injected intramuscularly is intended to be administer by inhalation for the treatment of pulmonary tuberculosis.In order to increase the drug residence time in the lung, capreomycin hydrophobicity was enhanced by substituting sulfate with oleate, thus obtaining capreomycin oleate (CO). The generation of a more hydrophobic ion-pair allows the reduction of the drug solubilisation in the bronchoalveolar fluids as well as its systemic absorption.The aim of the present study was to quantify CO in an in-house prepared drug formulation for inhalation. In this regard, a Hydrophilic Liquid Interaction Chromatography (HILIC) method was optimized with acetonitrile (ACN)/water containing eluents and a diol-type stationary phase. The optimal eluent composition [ACN/water-80/20 (v/v), 20 mM ammonium formate, 3.0 w s p H ] produced a good separation (α equal to 1.15) between the two main peaks. The developed HILIC method succeeded in the quantitative assay of CO in the drug formulation and was fully validated. Very good precision and accuracy in the short- and long-period along with appreciably low LOD and LOQ values (respectively 1.75 and 5.25 μg/mL) turned out.
Keywords: Capreomycin oleate; Dry powder for inhalation; Hydrophilic interaction liquid chromatography; Method validation;

Screening of NOS activity and selectivity of newly synthesized acetamidines using RP-HPLC by Marialuigia Fantacuzzi; Cristina Maccallini; Mauro Di Matteo; Alessandra Ammazzalorso; Isabella Bruno; Barbara De Filippis; Letizia Giampietro; Adriano Mollica; Rosa Amoroso (419-424).
Display OmittedNitric Oxide Synthase (NOS) inhibitors could play a powerful role in inflammatory and neurodegenerative diseases. In this work, novel acetamidine derivatives of NOS were synthesized and the inhibitor activity was evalued. To screen the activity and selectivity, the l-citrulline residue, after the enzymatic NOS assay, was derivatized with o-phthaldialdehyde/N-acetyl cysteine (OPA/NAC) and then evaluated by RP-HPLC method with fluorescence detection.All compounds did not affect the activity of endothelial and neuronal isoforms, while nine of them possessed a percentage of iNOS activity at 10 μM lower than 50%, and were selected for IC50 evaluation. Among them, a compound emerged as a very potent (IC50 of 53 nM) and selective iNOS inhibitor.
Keywords: High-performance liquid chromatography; Nitric Oxide Synthase inhibitor; Acetamidine; Fluorescence detection; OPA/NAC derivatization;