Journal of Chromatography B (v.940, #C)

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25 °C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis–Menten kinetics showed that the K m values of UTL-5g were 2.07 mM with PLE and 0.37 mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA.
Keywords: Esterase; Enzymatic products; HPLC approach; Peptide bond; Metabolite; Michaelis–Menten kinetics;

A novel derivatization reagent, (S)-1-(4-dimethylaminophenylcarbonyl)-3-aminopyrrolidine (1-DAPAP), was developed for increasing the detection sensitivity and enantiomeric separation of chiral carboxylic acids by liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI-MS/MS). 1-DAPAP reacted with carboxylic acids at room temperature within 5 min in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The epimerization (racemization) during the derivatization reaction was negligible. The resulting derivatives were highly responsive during the ESI-MS operating in the positive-ion mode and gave a characteristic product ion during the MS/MS, which enabled the sensitive detection using selected reaction monitoring; the detection responses of the 1-DAPAP-derivatives were increased by 10–1100-fold over the intact carboxylic acids and the limits of detection ranged from 0.97 and 5.2 fmol on the column. The 1-DAPAP-derivatization was also effective for the enantiomeric separation of chiral carboxylic acids; the resolution values were 1.2–4.3 for the evaluated carboxylic acids. The derivatization procedure was successfully applied to biological sample analyses; the derivatization followed by LC/ESI-MS/MS enabled the separation and detection of trace amounts of ibuprofen and naproxen in human saliva with a simple pretreatment and small sample volume.
Keywords: Derivatization reagent; Chiral carboxylic acid; Enantiomeric separation; Increasing sensitivity; LC/ESI-MS/MS;

Liquid chromatography tandem mass spectrometry method was developed and validated to confirm of resorcylic acid lactones: zeranol, taleranol, zearalanone, zearalenone, α and β-zearalenol and stilbenes in muscle tissue. The compounds were analyzed by LC–MS/MS QTrap 5500 apparatus in negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 (150 mm × 2.1 mm, 2.7 μm) column was achieved at 45 °C using isocratic elution of mobile phase – methanol/water (65:35, v/v). For the treatment of tissue samples prior to analysis, QuEChERS method was applied based on the extraction of analytes from muscle samples with ethyl acetate, separation of the aqueous and organic phases with application of magnesium sulphate and sodium acetate, the purification of the extract obtained by dispersive SPE with the use of sorbent C18, PSA and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. Good recoveries were obtained (from 83% to 115%) as well as acceptable within-lab reproducibility (<22%). The values of the decision limit CCα and the detection capability CCβ for individual compounds are found to be below the recommended concentration set at 1 μg kg−1 and not exceed 0.23 μg kg−1 and 0.39 μg kg−1, respectively. The elaborated method meets the criteria for confirmatory methods and is used in the official control of hormones.
Keywords: Stilbenes; RALs; QuEChERS; Liquid chromatography; Mass spectrometry;

Androgen glucuronides analysis by liquid chromatography tandem-mass spectrometry: Could it raise new perspectives in the diagnostic field of hormone-dependent malignancies? by Eleni Kalogera; Constantinos Pistos; Xeni Provatopoulou; Sotirios Athanaselis; Chara Spiliopoulou; Antonia Gounaris (24-34).
Breast and prostate constitute organs of intense steroidogenic activity. Clinical and epidemiologic data provide strong evidence on the influence of androgens and estrogens on the risk of typical hormone-dependent malignancies, like breast and prostate cancer. Recent studies have focused on the role of androgen metabolites in regulating androgen concentrations in hormone-sensitive tissues. Steroid glucuronidation has been suggested to have a prominent role in controlling the levels and the biological activity of unconjugated androgens. It is well-established that serum levels of androgen glucuronides reflect androgen metabolism in androgen-sensitive tissues. Quantitative analysis of androgen metabolites in blood specimens is the only minimally invasive approach permitting an accurate estimate of the total pool of androgens. During the past years, androgen glucuronides analysis most often involved radioimmunoassays (RIA) or direct immunoassays, both methods bearing serious limitations. However, recent impressive technical advances in mass spectrometry, and particularly in high performance liquid chromatography coupled with mass spectrometry (LC–MS/MS), have overcome these drawbacks enabling the simultaneous, quantitative analysis of multiple steroids even at low concentrations. Blood androgen profiling by LC–MS/MS, a robust and reliable technique of high selectivity, sensitivity, specificity, precision and accuracy emerges as a promising new approach in the study of human pathology. The present review offers a contemporary insight in androgen glucuronides profiling through the application of LC–MS/MS, highlighting new perspectives in the study of steroids and their implication in hormone-dependent malignancies.
Keywords: Androgen glucuronides; Liquid chromatography; Mass spectrometry; Hormone-dependent malignancies; Quantitative analysis; Review;

Development of a high performance liquid chromatography method for quantification of isomers β-caryophyllene and α-humulene in copaiba oleoresin using the Box-Behnken design by Vinícius Raphael de Almeida Borges; Ana Ferreira Ribeiro; Carina de Souza Anselmo; Lúcio Mendes Cabral; Valéria Pereira de Sousa (35-41).
The sesquiterpene isomers, β-Cariofileno (CAR) and α-Humuleno (HUM) are the primary constituents of the copaiba oleoresin species. These natural products are primarily used by the Amazonian population and marketed as phytotherapies and cosmetics. The aim of this study was to develop and validate a method that simultaneously assays the isomers present in copaiba oleoresins by high performance liquid chromatography using the Box-Behnken design. After preliminary studies, the reverse phase chromatographic system was selected using a cyano column and a mobile phase consisting of acetonitrile and phosphate buffer. The Box-Behnken design was applied at three levels and with four independent variables: flow rate (X 1), gradient slope time (X 2), proportion of organic compounds at the end of the gradient (X 3) and at the beginning of the gradient (X 4). Also, the responses of the dependent variables: CAR retention time (Y 1) and the resolution between the CAR and HUM peaks (Y 2) was assessed. The mathematical model obtained from the regression results was satisfactory (R 2  > 0.98, n  = 27) and showed a quadratic relationship where the effects of interactions between the variables, was observed by response surface graphs. The simultaneous optimization method was used to establish the best compromise of the resolution between the CAR and HUM isomers while adjusting the retention time of CAR. This method was successfully optimized by BBD obtaining chromatographic peaks with good symmetry, resolution and separation efficiency. The validation of the developed method confirmed its specificity, precision, accuracy and linearity in the range of 5.0–11.0 and 0.4–1.0 μg/mL for CAR and HUM, respectively, and is considered suitable for routine applications which assure quality control.
Keywords: Box Benhken; HPLC; β-Caryophyllene; α-Humulene; Copaiba oleoresin; DOE;

Liquid chromatography–mass spectrometric determination of rufinamide in low volume plasma samples by Zsolt Gáll; Szende Vancea; Maria T. Dogaru; Tibor Szilágyi (42-46).
Quantification of rufinamide in plasma was achieved using a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method. The chromatographic separation was achieved on a reversed phase column (Zorbax SB-C18 100 mm × 3 mm, 3.5 μm) under isocratic conditions. The mobile phase consisted of a mixture of water containing 0.1% formic acid and methanol (50:50, v/v). The mass spectrometric detection of the analyte was in multiple reaction monitoring mode (MRM) using an electrospray positive ionization (ESI positive). The monitored ions were 127  m/z derived from 239  m/z rufinamide and 108  m/z derived from 251  m/z the internal standard (lacosamide). Protein precipitation with methanol was applied for sample preparation using only 50 μl aliquots. The concentration range was 40–2000 ng/ml for rufinamide in plasma. The limit of detection was 1.25 ng/ml and the lower limit of quantification was established at 5 ng/ml rufinamide concentration. Selectivity and matrix effect was verified using individual human, rat and rabbit plasma samples. Short-term, post-preparative and freeze–thaw stability was also investigated. The proposed method provides accuracy, precision and high-throughput (short runtime 4.5 min) for quantitative determination of rufinamide in plasma. This is the first reported liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for analysis of rufinamide from low volume plasma samples. The LC–MS/MS method was validated according to the current official guidelines and can be applied to accurately measure rufinamide level of large number of plasma samples from clinical studies or therapeutic drug monitoring.
Keywords: Antiepileptic drug; Rufinamide; Plasma; Liquid chromatography–tandem mass spectrometry; Validation;

Stereoselective analysis of nebivolol isomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry: Application in pharmacokinetics by Daniel Valente Neves; Carolina Pinto Vieira; Eduardo Barbosa Coelho; Maria Paula Marques; Vera Lucia Lanchote (47-52).
Nebivolol is available for clinical use as a racemic mixture. Isomer d-nebivolol (SRRR) is a β1 adrenergic receptor blocker and its antipode, l-nebivolol (RSSS) is responsible for endothelium-dependent NO liberation. This report describes the development and validation of a method of analysis of nebivolol isomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). Nebivolol isomers were extracted from 2 mL aliquots of plasma spiked with tramadol as internal standard, alkalinized and added of sodium chloride and diisopropyl ether:dichloromethane (70:30, v/v). Nebivolol isomers were resolved on a Chirobiotic® V column using methanol:acetic acid:diethylamine (100:0.15:0.05, v/v/v) as mobile phase. Protonated ion and respective ion product were monitored in transitions 406 > 151 for nebivolol and 264 > 58 for internal standard tramadol. There was no racemization of nebivolol isomers during the procedures of sample preparation and chromatographic analysis and matrix effect was absent. Analysis of nebivolol isomers showed linearity for plasma concentrations of 25–2500 pg/mL of each isomer. The quantification limit was 25 pg of each isomer/mL of plasma. Variation coefficients and inaccuracy calculated in precision and accuracy determinations were lower than 15%. Nebivolol was stable in human plasma after three successive cycles of freezing and thawing, during 4 h at room temperature and after processing during 12 h in the auto sampler at 5 °C showing deviation values lower than 15%. The method was applied in a study of the kinetic disposition of nebivolol in plasma samples collected until 48 h after administration of an oral single dose of 10 mg of racemic nebivolol hydrochloride to a patient with systemic arterial hypertension. The clinical study demonstrated that the nebivolol pharmacokinetics is stereoselective. Isomer l-nebivolol showed higher AUC0–∞ (9.4 ng/h/mL vs. 4.7 ng/h/mL) and smaller apparent clearance (Cl/f) (531.8 L/h vs. 1304.4 L/h) when compared to antipode d-nebivolol.
Keywords: Nebivolol; Isomers; LC–MS/MS; Pharmacokinetics; Validation; Systemic arterial hypertension;

There is increasing recognition that the host response to critical illness includes derangement of multiple amino acid pathways, including amino acids (AAs) central to metabolism and immune, endothelial and neurological function. To characterise concentration changes of these plasma amino acid we report the development and validation of a method for the quantification of AAs in small volumes of plasma (50 μL) using HPLC with simultaneous UV and fluorescence (FL) detection. Protein precipitation and pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is followed by reversed phase HPLC separation. Calibration curves were built with norleucine as an internal standard. Thirty-three (including the 20 proteinogenic) AAs, were selected as standards and their corresponding concentrations in the plasma of healthy human controls and patients with severe falciparum malaria were quantified. This method enables the detection of perturbations in arginine metabolism, aromatic amino acid pathways, methionine transsulfuration and transmethylation pathways and other metabolic pathways.
Keywords: Amino acids; Malaria; Critical illness; HPLC;

MCM-41 solid phase membrane tip extraction combined with liquid chromatography for the determination of non-steroidal anti-inflammatory drugs in human urine by Sazlinda Kamaruzaman; Mohd Marsin Sanagi; Salasiah Endud; Wan Aini Wan Ibrahim; Noorfatimah Yahaya (59-65).
Mesoporous silica material, MCM-41, was utilized for the first time as an adsorbent in solid phase membrane tip extraction (SPMTE) of non-steroidal anti-inflammatory drugs (NSAIDs) in urine prior to high performance liquid chromatography-ultraviolet (HPLC-UV) analysis. The prepared MCM-41 material was enclosed in a polypropylene membrane tip and used as an adsorbent in SPMTE. Four NSAIDs namely ketoprofen, diclofenac, mefenamic acid and naproxen were selected as model analytes. Several important parameters, such as conditioning solvent, sample pH, salting-out effect, sample volume, extraction time, desorption solvent and desorption time were optimized. Under the optimum extraction conditions, the MCM-41-SPMTE method showed good linearity in the range of 0.01–10 μg/mL with excellent correlation coefficients (r  = 0.9977–0.9995), acceptable RSDs (0.4–9.4%, n  = 3), good limits of detection (5.7–10.6 μg/L) and relative recoveries (81.4–108.1%). The developed method showed a good tolerance to biological sample matrices.
Keywords: Mesoporous silica; Solid phase membrane tip extraction; Non-steroidal anti-inflammatory drugs; Urine;

Development and validation of novel, general liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of amoxicillin (AMOX), cefatoxime (CEF), ciprofloxacin (CIP), daptomycin (DAPTO), fluconazole (FLU), gentamicin (GEN), clindamycin (KLI), linezolid (LIN), metronidazole (MET), moxifloxacin (MOXI) in human whole blood are described. Samples were prepared on solid phase microextraction way with the use of polymeric sorption coatings with molecular imprints and analyzed using a gradient separation over an ACE C18-column (4.6 mm × 150 mm, 3 μm) with isocratic elution. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (0.1% formic acid or 5 mM ammonium acetate) at a flow 0.4 ml/min. The chromatographic run time was kept less than 9 min. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 7.3%, while the accuracy was within ±8.4%. The mean recovery of all the analytes ranged from 65.0 to 83.0%. This method was successfully applied to clinical samples from patients with clinically diagnosed bacterial infections process.
Keywords: Antibiotic drugs; Liquid chromatography–mass spectrometry; Molecular imprinted polymers; Solid phase microextraction; Whole human blood;

In this study, metabolites in the urine samples of rats orally administered with acteoside, a phenylethanoid glycoside compound, were detected and identified using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) combined with an automated MSE technique. Up to 35 metabolites (19 metabolites of the parent drug and 16 metabolites of the degradation products) were observed, including processes of oxidization, glucuronidation, sulfation, and methyl conjugation. According to the metabolic pathways, acteoside mainly functioned as a prodrug and underwent hydrolysis before being absorbed into the blood. The degradation products, especially caffeic acid and hydroxytyrosol, were involved in further metabolism which was responsible for the low oral bioavailability but obvious pharmacological activities of acteoside. In summary, this work provided valuable information on acteoside metabolism through the rapid and reliable UPLC/ESI-QTOF-MS technique, which could be widely used for the investigation of natural product metabolites.
Keywords: Acteoside; Prodrug; Metabolite identification; UPLC/ESI-QTOF-MS; MSE technique;

Eucommia ulmoides bark has long been known as tonic herb, however, the systematic biochemical fingerprint analysis and corresponding structural identification are inadequate so far. Here we describe on-line surface plasmon resonance-high performance liquid chromatography–tandem mass spectrometry (SPR-HPLC–MS/MS) to fish out and identify human serum albumin (HSA) binders from E. ulmoides bark. A total of 22 HSA binders, including 4 iridoids, 11 lignans, 3 flavonoids and 4 phenolic acids, were identified or tentatively characterized based on their retention times, UV spectra and MS/MS spectrum with references or literature data, among which, geniposidic acid, geniposide and chlorogenic acid were the predominant HSA binders. In addition, the major characteristic fragmentation pathways of iridoids and lignans were investigated. The higher reusability and stability of the immobilized HSA allow the method cost-effective and high-throughput. Compared with conventional HPLC–MS/MS for chemical fingerprint analysis, SPR-HPLC–MS/MS is simple and easy to provide biochemical fingerprint, moreover, it is easy to identify low-content HSA binders because of the sample clean-up/concentration procedure.
Keywords: SPR; HPLC–MS/MS; HSA; Eucommia ulmoides bark; Iridoid; Lignan;

Profiling analysis of biogenic amines and their acidic metabolites in mouse brain tissue using gas chromatography–tandem mass spectrometry by Joo Yeon Hong; Na Hyun Park; Myung Sook Oh; Hye Suk Lee; Heesoo Pyo; Jongki Hong (94-103).
The profiling analysis of biogenic amines, including catecholamines and serotonin, and their metabolites in mouse brain tissue provides an important key to understanding their roles in the body and the possibility of simple and accurate diagnosis of neural diseases. A novel approach in the analysis of biogenic amines and their acidic metabolites in brain tissue using gas chromatography–tandem mass spectrometry (GC–MS/MS) is presented. Biogenic amines and their acidic metabolites in brain tissue were effectively separated using a mixed-cation-exchange solid-phase extraction (MCX-SPE) cartridge with a variation in the composition of the SPE elution solvents. A selective derivatization with hexamethyldisilazane (HMDS) and N-methyl-bis-heptafluorobutyramide (MBHFBA) was used to increase the detection sensitivity and to prevent the formation of any side-products. The identification and quantification of the target analytes were performed by gas chromatography triple quadrupole mass spectrometry (GC–MS/MS) using multiple ion reaction monitoring (MRM) mode. The overall recovery yields of the biogenic amines and their metabolites were above 87.5% at 10 ng/g and 92.4% at 100 ng/g of spiking concentration range. The isotopic-labeled internal standards were used for the precise quantification of bioamines and their metabolites. The calibration curves for the biogenic amines and their metabolites obtained through GC–MS/MS were linear (r 2  > 0.995) over the concentration range of 1 (2 or 3)–200 ng/mL. The present method was reproducible (relative standard deviation range 0.6–9.3%) and accurate (range 85.4–107.9%), with LLOQs of 0.71–3.69 ng/mL. The developed method was successfully applied to the determination and quantification of bioamines and their metabolites in rat brain tissue samples.
Keywords: Biogenic amines; Acidic metabolites; Mouse brain tissues; Selective derivatization; GC–MS/MS;

Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties by Chandrasekhar Gurramkonda; Maria Zahid; Satish Kumar Nemani; Ahmad Adnan; Satheesh Kumar Gudi; Navin Khanna; Thomas Ebensen; Heinrich Lünsdorf; Carlos A. Guzmán; Ursula Rinas (104-111).
Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600 bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.
Keywords: Hepatitis B surface antigen virus-like particles; Aerosil-380; Ion-exchange chromatography; Ultracentrifugation; Size-exclusion chromatography; Electron microscopy; Vaccine;

The room temperature ionic liquid isopropylammonium formate (IPAF) is studied as a reversed phase HPLC mobile phase modifier for separation of native proteins using a polymeric column and the protein stability is compared to that using acetonitrile (MeCN) as the standard organic mobile phase modifier. A variety of important proteins with different numbers of subunits are investigated, including non-subunit proteins: albumin, and amyloglucosidase (AMY); a two subunit protein: thyroglobulin (THY); and four subunit proteins: glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH). A significant enhancement in protein stability is observed in the chromatograms upon using IPAF as a mobile phase modifier. The first sharper peak at about 2 min represented protein in primarily the native form and a second broader peak more retained at about 5–6 min represented substantially denatured or possibly aggregated protein. The investigated proteins (except LDH) could maintain the native form within up to 50% IPAF, while a mobile phase, with as low as 10% MeCN, induced protein denaturation. The assay for pyruvate using LDH has further shown that enzymatic activity can be maintained up to 30% IPAF in water in contrast to no activity using 30% MeCN.
Keywords: Ionic liquid; High performance liquid chromatography; Protein stability; Mobile phase modifier;

Olaparib is an inhibitor of poly ADP ribose polymerase 1 (PARP-1). Phase I and II trials showed promising results of olaparib against tumours in BRCA mutation carriers. Currently an increasing number of clinical trials with olaparib in combination with other compounds or radiotherapy are conducted. To support these clinical trials an LC–MS/MS method was developed and validated for the quantification of olaparib in human plasma. Human plasma samples were collected in the clinic and stored at nominally −20 °C. Olaparib was isolated from plasma by liquid–liquid extraction, separated on a C18 column with gradient elution and analyzed with triple quadrupole mass spectrometry in positive ion mode. A deuterated isotope was used as internal standard for the quantification. The assay, ranging from 10 to 5000 ng/mL, was linear with correlation coefficients (r 2) of 0.9994 or better. The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±7.6% of nominal and inter-assay and intra-assay precision ≤9.3% at the lower limit of quantification and ≤5.7% at the other concentration levels tested. All results were within the acceptance criteria of the US FDA and the latest EMA guidelines for method validation. A quantitative method was developed and validated for the quantification of olaparib in human plasma. The method could successfully be applied for the pharmacokinetic quantification of olaparib in cancer patients treated with olaparib.
Keywords: Olaparib; PARP-inhibitor; LC–MS/MS; GLP; Validation; Human plasma;

A hyphenated procedure of heat-reflux and ultrasound-assisted extraction (HUAE), and an accurate high-performance liquid chromatographic (HPLC) method were developed for the determination of apigenin, baicalin and luteolin content in Scutellaria barbara D. Don. The suitable HUAE conditions for the extraction of target compounds from the herb were identified as an ultrasonic frequency of 40 kHz, power of 185 W, duty cycle of 75% (intermittent sonication), mean particle size of 0.355 mm, extraction temperature of 50 °C, ratio of solvent to raw material of 12:1 (mL/g), ethanol concentration of 60% (v/v), extraction time of 30 min and three cycles. Compared with a traditional heat-reflux extraction method, the proposed method reduced the extraction time, extraction temperature and solvent consumption. Also, this HUAE method achieved superior apigenin, baicalin and luteolin yields. Furthermore, the developed HUAE–HPLC method was applied successfully for the simultaneous evaluation of three bioactive compounds in five samples of S. barbara D. Don obtained from different geographical regions. These results clearly demonstrated that the combined HUAE–HPLC process is feasible in the future commercialized manufacture of this highly valuable Chinese herbal medicine.
Keywords: Scutellaria barbata D. Don; Ultrasound-assisted extraction; HPLC; Apigenin; Baicalin; Luteolin;

Isolation, cytotoxicity evaluation and HPLC-quantification of the chemical constituents from Artemisia amygdalina Decne by Shabir H. Lone; Khursheed A. Bhat; Syed Naseer; Rafiq A. Rather; Mohd A. Khuroo; Sheikh A. Tasduq (135-141).
The hexane extracts of both shoot and root parts of Artemisia amygdalina Decne displayed potent cytotoxic effects. Phytochemical analysis of these active extracts led to the isolation of six cytotoxic constituents, viz., Ergostadien-3β-ol (1), ludartin (2), 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (3) (from shoot) and trans-matricaria ester (4), diacetylenic spiroenol ether (5) and cis-matricaria ester (6) (from root) for the first time from this plant. The constituents were identified using spectral techniques in the light of literature. Sulphorhodamine B cytotoxicity screening of the isolated constituents was carried out against four human cancer cell lines including Lung (A-549), Leukaemia (THP-1), Prostate (PC-3) and Colon (HCT-116) cell lines. Ludartin (2) exhibited the highest cytotoxicity with IC50 values of 7.4 μM, 3.1 μM, 7.5 μM and 6.9 μM against Lung (A-549), Leukaemia (THP-1), Prostate (PC-3), Colon (HCT-116) cancer cell lines respectively. To test against in vitro skin cancer models [human dermal fibroblasts (CRL-1635)] all the isolates were further subjected to 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) cytotoxicity screening. Ludartin (2) being highly cytotoxic was again evaluated against mouse melanoma (B16F10) and human epidermoid carcinoma (A-431) cells by MTT assay displaying IC50 values of 6.6 μM and 19.0 μM respectively. Finally a simple and reliable HPLC method was developed (RP-HPLC-DAD) and validated for the simultaneous quantification of these cytotoxic constituents in A. amygdalina Decne. Excellent specificity and high linearity for all the standard calibration curves having regression coefficients of the respective linear equations in the range of 0.9962–0.9999 was observed. Relative recovery rates varied between 98.37 ± 0.90 and 105.15 ± 1.74 with relative standard deviation less than 4%. Based on our results, the developed method features good quantification parameters, accuracy, precision and can serve as effective quality control method for standardisation of A. amygdalina Decne.
Keywords: Artemisia amygdalina Decne; Isolation; Ludartin; Cytotoxicity; HPLC;

A one-step derivatization and microextraction technique for the determination of amantadine in the human plasma and urine samples is presented. An appropriate mixture of methanol (disperser solvent), 1,2-dibromoethane (extraction solvent), and butylchloroformate (derivatization agent) is rapidly injected into samples. After centrifuging, the sedimented phase is analyzed by gas chromatography-flame ionization detection (GC-FID). The kind of extraction and disperser solvents and their volumes, amount of derivatization agent and reaction/extraction time which are effective in derivatization/dispersive liquid–liquid microextraction (DLLME) procedure are optimized. Under the optimal conditions, the enrichment factor (EF) of the target analyte was obtained to be 408 and 420, and limit of detection (LOD) 4.2 and 2.7 ng mL−1, in plasma and urine respectively. The linear range is 14–5000 and 8.7–5000 ng/mL for plasma and urine, respectively (squared correlation coefficient ≥ 0.990). The relative recoveries obtained for the spiked plasma and urine samples are between 72% and 93%. Moreover, the inter- and intra-day precisions are acceptable at all spiked concentrations (relative standard deviation <7%). Finally the method was successfully applied to determine amantadine in biological samples.
Keywords: Dispersive liquid–liquid microextraction; Derivatization; Amantadine; Gas chromatography;