Journal of Chromatography B (v.879, #21)

Surface energetics of bone mineral and synthetic hydroxyapatite using inverse gas chromatography by Bhushan B. Hole; D. Steven Keller; William M. Burry; James A. Schwarz (1847-1850).
Surface energy is one of the important factors that govern protein adhesion and cell attachment on biomaterial surfaces. Inverse gas chromatography (IGC) provides an excellent method to measure the surface energetics of rough and porous biosurfaces. In this study IGC was used to characterize and compare the surface energetics of synthetic and biological hydroxyapatites (natural bone mineral). IGC experiments were performed on three samples: synthetic hydroxyapatites with two levels of purity (99% and 90%) and natural biological hydroxyapatite obtained from bovine trabecular bone. The Lifshitz–Van der Waals component of the surface free energy ( γ S L W ) and specific interaction parameter (ɛ π) were determined by using homologous series of n-alkanes and alkenes as IGC probe molecules, respectively. The synthetic hydroxyapatite had values of γ S L W of 33.4 mJ m−2 at 99% purity and 53.3 mJ m−2 at 90% purity. Biological hydroxyapatite had a value of γ S L W of 45.7 mJ m−2. For the synthetic hydroxyapatite, the values of π-bond specific interaction parameters, ɛ π, were 0.95 mJ (99%) and 3.01 mJ (90%). The biological hydroxyapatite sample had a value of 2.44 mJ for ɛ π. The results suggest that, as compared to the synthetic compounds, the biological apatite has considerable surface heterogeneity, either chemical (impurities) or structural suggesting a scaffold surface that is more conducive of protein adhesion and cell attachment.
Keywords: Inverse gas chromatography; Hydroxyapatite coating; Biomaterial; Surface energy; Bone; Biological scaffolds;

Liquid chromatography–tandem mass spectrometric assay for the PARP-1 inhibitor olaparib in combination with the nitrogen mustard melphalan in human plasma by Rolf W. Sparidans; Irene Martens; Liselot B.J. Valkenburg-van Iersel; Jan den Hartigh; Jan H.M. Schellens; Jos H. Beijnen (1851-1856).
A bioanalytical assay for the new poly(ADP-ribose) polymerase-1 inhibitor olaparib in combination with melphalan was developed and validated. For the quantitative assay, human plasma samples were pre-treated on ice using protein precipitation with 2% (v/v) acetic acid in acetonitrile containing erlotinib and melphalan-d8 as internal standards. The extract was diluted with water and injected into the chromatographic system. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with an isocratic elution using 0.01% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10–5000 ng/ml calibration range for both drugs. The lowest level of this range corresponded to the lower limit of quantification. Within day precisions were 3.0–9.3%, between day precisions 6.0–9.8% and accuracies were between 101 and 110% for the whole calibration range. After validation the assay was used to assess the pharmacokinetics of olaparib in a patient with metastatic breast carcinoma. In addition, systemic exposure of melphalan was monitored in patients subjected to isolated hepatic perfusion with this drug. Both applications show that the new assay can be applied for human pharmacokinetic studies for both drugs.
Keywords: Olaparib; Melphalan; PARP inhibitor; LC–MS/MS; Human plasma;

A transglycosylation reaction between an alkyl polyglycoside and α-cyclodextrin catalysed by cyclodextrin glycosyltransferase (CGTase) from Bacillus macerans was investigated. The reaction products were identified by comparison with standards generated by CGTase catalysed modification of pure alkyl glycosides using HPLC–ELSD and –MS analysis. The main products were alkyl glucopyranosides (substrates present in the alkyl polyglycoside) glycosylated with 6 (primary coupling products) or 12 (secondary coupling products) glucose residues. Both α and β anomers were glycosylated.
Keywords: Cyclodextrin glycosyltransferase; CGTase; Alkyl polyglycoside; APG; HPLC–MS;

Simultaneous analysis of synthetic musks and triclosan in human breast milk by gas chromatography tandem mass spectrometry by Hao Wang; Jing Zhang; Fudie Gao; Yi Yang; Hejun Duan; Yongning Wu; Jean-Daniel Berset; Bing Shao (1861-1869).
A comprehensive method was developed for the simultaneous analysis in human breast milk of 12 synthetic musks, five nitro musks, six polycyclic muks and one macrocyclic musk; as well as one musk metabolite and triclosan. The target analytes were freeze dried and extracted using the accelerated solvent extraction (ASE) procedure. The extracts were further purified by gel permeation chromatography (GPC) and florisil solid-phase extraction (SPE) and then analyzed by gas chromatography tandem mass spectrometry (GC–MS/MS). Recoveries of the analytes based on the isotopic internal standard correction ranged from 82.4% to 112%, with relative standard derivations less than 20%. The method quantification limits (MQLs) were 0.6–5.4 ng/g lipid. The analytes were detected in human breast milk samples and ranged from 11.7 to 308.6 ng/g lipid.
Keywords: Synthetic musks; Triclosan; ASE; GC–MS/MS; Human breast milk;

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 μL of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n  = 9). The linear range was 7.5–850 μg L−1 with correlation coefficient (r 2  = 0.999), detection limits was 2.5 μg L−1 and the LOQ was 7.5 μg L−1. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae.
Keywords: Hollow-fiber membrane; Liquid phase microextraction; Valerenic acid; HPLC/UV; Valerian root;

Assay for determination of daunorubicin in cancer cells with multidrug resistance phenotype by Petra Krumpochova; Andrea Kocurova; Petr Dolezel; Petr Mlejnek (1875-1880).
A sensitive assay for direct determination of intracellular level of daunorubicin (DRN) in resistant leukemia cells with overexpressed P-glycoprotein has been developed. This assay is based on a rapid separation of cells from media and fast cut-off of DRN transportation by centrifugation of cells through a layer of silicone oil. Cell pellets were extracted using 1% (v/v) formic acid in 50% (v/v) ethanol in water. The cell extracts were subsequently analysed by liquid chromatography (HPLC) coupled a low-energy collision tandem mass spectrometer equipped with an electrospray ionization source (ESI–CID–MS/MS) operated in the multiple-reaction monitoring (MRM) mode. Calibration curve was linear from 0.4 to 250 nM with correlation coefficient (r 2) better than 0.998. The limit of quantitation (LOQ) was 0.4 nM. The assay has been successfully applied to a determination of intracellular content of daunorubicin in sensitive K562 and resistant K562/Dox and K562/HHT300 cells.
Keywords: Intracellular drug level; K562 cells; Daunomycin; P-glycoprotein; ABCB1; Silicone oil;

Application of the response surface methodology for optimization of whey protein partitioning in PEG/phosphate aqueous two-phase system by Lizzy Ayra Pereira Alcântara; Luis Antonio Minim; Valéria Paula Rodrigues Minim; Renata Cristina Ferreira Bonomo; Luis Henrique Mendes da Silva; Maria do Carmo Hespanhol da Silva (1881-1885).
In order to develop a new strategy for β-lactoglobulin (β-lg) removal from whey protein, partitioning of α-lactalbumin (α-la), β-lg and glycomacropeptide (Gmp) was studied using aqueous two phase systems (ATPS). A system composed of 13% (w/w) polyethylene glycol (PEG, average molar mass 2000 g/mol) and 13% (w/w) potassium phosphate was used at 25 °C. A central composite rotatable design (CCRD) associated to the response surface methodology (RSM) was applied to investigate the effects of NaCl concentration and pH on the partition of these proteins. It was found that α-la and Gmp partitioned to the top phase rich in PEG, whereas β-lg partitioned to the bottom phase rich in salt. According to the RSM, optimal conditions for β-lg removal where found where pH was equal to 6.7 and salt concentration was 0.35 mol/L. Under these conditions, the partition coefficient K α was 0.48 and K Gmp was 0.92. On the other hand, the partition coefficient K β was only 0.01. In such conditions β-lg preferentially concentrates in the bottom phase, while the top phase exclusively contains the proteins α-la and Gmp. Fractionation of the proteins from fresh whey was performed in a three stage cross-flow extraction system. The extraction yield for β-lg in the bottom phase was 97.3%, while the yields for α-la and Gmp in the top phase were 81.1% and 97.8%, respectively.
Keywords: Aqueous two phase systems; Partitioning; α-Lactalbumin; β-Lactoglobulin; Glycomacropeptide;

Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS by Flaubert Mbeunkui; Mary H. Grace; Carmen Lategan; Peter J. Smith; Ilya Raskin; Mary Ann Lila (1886-1892).
The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes–ethyl acetate–methanol–water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), 1H nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity.
Keywords: Centrifugal partition chromatography; N-alkylamides; Antiplasmodial activity;

The aim of this study is to develop a standardized LC–MS/MS method for accurate measurement of desmosine (DES) and isodesmosine (IDS) in all body fluids as biomarkers for in vivo degradation of matrix tissue elastin in man and animals. A reproducible three-step analytical procedure: (1) sample hydrolysis in 6 N HCl, (2) SPE by a CF1 cartridge with addition of acetylated pyridinoline as internal standard (IS), and (3) LC/MSMS analysis by SRM monitoring of transition ions; DES or IDS (m/z 526–481 + 397) and IS (m/z 471–128) was developed. The method achieves accurate measurements of DES/IDS in accessible body fluids (i.e. urine, plasma, and sputum). LOQ of DES/IDS in body fluids is 0.1 ng/ml. The % recoveries and reproducibility from urine, plasma, and sputum samples are above 99 ± 8% (n  = 3), 94 ± 9% (n  = 3) and 87 ± 11% (n  = 3), with imprecision 8%, 9% and 10%, respectively. The proposed method was applied to measure DES/IDS in body fluids of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Total DES/IDS in sputum and plasma is increased over normal controls along with the free DES/IDS in urine in patients. DES/IDS can be used to study the course of COPD and the response to therapy. This practical and reliable LC–MS/MS method is proposed as a standardized method to measure DES and IDS in body fluids. This method can have wide application for investigating diseases which involve elastic tissue degradation.
Keywords: LC–MS; Elastin degradation; Biomarker; Chronic obstructive pulmonary disease;

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum–ethyl acetate–methanol–water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2 mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3 mg of cryptotanshinone, 16.2 mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3 mg of miltirone were obtained from 400 mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.
Keywords: High-speed counter-current chromatography; Salvia miltiorrhiza Bunge; Tanshinone;

Exemestane, irreversible steroidal aromatase inhibitor, acts as a false substrate for aromatase enzyme and significantly lowers circulating estrogen concentrations in postmenopausal women with hormone-sensitive breast cancer. A sensitive bioanalytical method was developed and validated to study pharmacokinetics of exemestane. The method was based on liquid–liquid extraction of exemestane with methyl t-butyl ether followed by reversed-phase liquid chromatography. Positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode was applied for detection of exemestane. Anastrozole was used as internal standard. Calibration curve, fitted to 1/x 2 weighted linear regression model, was linear in the range of 0.1–40.0 ng/mL. Intra-run precision and accuracy were 1.80–3.17% and 103.4–111.5%, respectively. Inter-run precision and accuracy measured within 3 days were 3.37–4.19% and 101.8–109.6%, respectively. Extraction recoveries of exemestane and internal standard were 79.7–86.2% and 82.9–83.6%, respectively. The method was fully validated and may be applied to pharmacokinetic studies in humans after a single dose administration of 25 mg exemestane tablets.
Keywords: Exemestane; Anastrozole; Liquid chromatography/electrospray ionization tandem mass spectrometry; Human plasma; Pharmacokinetics; Liquid–liquid extraction;

A simple and rapid reverse polar ionic LC method was developed and validated for simultaneous separation and determination of mirtazapine, an antidepressant drug, and its main metabolite N-desmethyl mirtazapine using fluorescence and polarimetric detectors connected in series. The chromatographic separation was achieved on Chirobiotic V column packed with vancomycin as a stationary phase in an isocratic mode of elution of methanol:glacial acetic acid:anhydrous triethyl amine (100:0.2:0.1, v/v/v) as a mobile phase. The compounds were detected by their excitation at 290 nm and emission at 370 nm using fluorescence detector while the optical rotation (+/−) of the enantiomers was identified by polarimetric detector. The analytes were extracted from rat plasma by precipitation of proteins and the average yield was 88–111% for mirtazapine and 85–123% for N-desmethyl mirtazapine. The method was linear over the concentration range of 20–5000 ng/mL. The method was successfully applied on rat plasma spiked with the enantiomers of mirtazapine and N-desmethyl mirtazapine.
Keywords: Mirtazapine; Antidepressants; Metabolite; Vancomycin; Enantiomeric separation; Reverse polar ionic LC;

Theory-guided efficient strategy to maximize speed and resolution in rapid gradient LC–MS/MS bioanalysis by Jian Wang; Jingpin Jia; Anne Aubry; Mark Arnold; Mohammed Jemal (1917-1926).
Reversed-phase gradient LC–MS/MS bioanalytical methods of 5–100% organic solvent in a 1–3 min gradient time are common in today's bioanalytical laboratory. The goal of this work was to develop a theory-guided systematic strategy for maximizing resolution and speed in rapid gradient LC–MS/MS bioanalysis. We studied the effect of gradient time (t G ), initial and final eluent strength (% B  = % organic), and flow rate (F) on the separation of multiple critical pairs (R s ) and peak capacity (n c ) in a gradient elution of a mixture of five structurally related compounds. By optimizing the gradient time t G , the initial and final percentages of the organic component of the mobile phase, comparable resolution and peak capacity could be achieved in a shorter run time. More importantly, we demonstrated that higher flow rates improved resolution, peak capacity and reduced run time in rapid gradient separations on a 5 μm particle column. A straightforward mathematical explanation of the phenomenon was provided applying basic resolution equations in gradient elution theory. A systematic approach to execute a rapid gradient LC–MS/MS bioanalytical method to shorten run time and improve resolution is proposed, taking into consideration not only the analytes of interest but also potential matrix effects from the dosing vehicle and biological matrix.
Keywords: LC–MS/MS bioanalysis; Rapid gradient elution; Flow rate; Resolution; Peak capacity;

Ultrafiltration of human plasma in combination with LC–MS/MS has been increasingly used in the quantitative analysis of the free fraction of drug candidates for PK/efficacy assessment. In addition to controlling the pre-incubation and centrifugation temperatures, some important factors that must be investigated and addressed include: (1) possible nonspecific binding, (2) possible impact of freeze/thaw cycles of plasma samples and extended storage of plasma samples at room temperature on the analyte recovery prior to ultrafiltration, and (3) identification of the appropriate assay dynamic range to avoid unnecessary dilutions. These factors were explored in the development and validation of a robust LC–MS/MS assay for the quantitative analysis of unbound vadimezan (ASA404) in human plasma. First, to mimic human physiological conditions, all plasma samples were incubated at ∼37 °C for a minimum of 30 min after thawing and prior to centrifugation to obtain the ultrafiltrate. Second, by passing the calibration standards and QC samples in plasma ultrafiltrate through the ultrafiltration membrane, the observed non-specific binding of the analyte due to the membrane was corrected. Third, the effects of multiple freeze/thaw cycles and/or storage at room temperature for various periods (4, 8, 16 and 24 h) were evaluated to determine the impact on analyte concentrations in the ultrafiltrate from the plasma QC samples. Fourth, the appropriate dynamic range was established to accommodate the expected incurred sample free analyte concentrations. The validated assay has a dynamic range of 30.0–30,000 ng/ml for ASA404 in human plasma ultrafiltrate using a sample volume of 30 μl. Quality control pools containing the analyte were prepared at concentrations of 30.0–22,500 ng/ml to cover the assay calibration range. The intra-assay and inter-assay precision and accuracy were ≤15% (CV) and within ±15% (bias) of the nominal values, respectively, for all measured QC concentrations, including the LLOQ. Freeze/thaw for up to three cycles of the plasma samples and/or the extended human plasma sample exposure to room temperature for up to 24 h were confirmed to have no impact on the assay results for the free analyte. The validated method was successfully implemented to support clinical studies for the compound.
Keywords: Vadimezan; ASA404; Ultrafiltration; Unbound analyte; Nonspecific binding; Stability; Incurred sample stability; Incurred sample reanalysis;

Investigation of competitive binding of ibuprofen and salicylic acid with serum albumin by affinity capillary electrophoresis by Zhu-Mei Li; Chuan-Wan Wei; Yintang Zhang; Di-Shan Wang; You-Nian Liu (1934-1938).
Ibuprofen and salicylic acid, two typical non-steroidal anti-inflammatory drugs, are used commonly as analgesic drug in clinical medicine and sometimes are co-administered. When the drugs are co-administered, the drug–drug interactions may occur, and can lead to alter the safety and efficacy of drugs, resulting in variations in drug response of the co-administered drugs. Affinity capillary electrophoresis (ACE) was employed to investigate the competitive binding of ibuprofen and salicylic acid on serum albumin. Mobility ratio, derivatives from mobility shift method, was used to deduce the binding constant (K b). The binding constants of ibuprofen with HSA are 2.97 × 106  M−1 and 7.07 × 104  M−1, respectively; while for salicylic acid, the binding constant is 5.99 × 104  M−1. The competitive binding of the two drugs was investigated by addition of excessive ibuprofen into the solutions containing constant concentrations of salicylic acid and serum albumin. The results confirmed that ibuprofen and salicylic acid have different high-affinity binding site, but share some low-affinity binding sites on the serum albumin; and ibuprofen is able to partially replace salicylic acid from the preformed binary complexes of serum albumin and salicylic acid.
Keywords: Affinity capillary electrophoresis; Competitive binding; Ibuprofen; Salicylic acid; Serum albumin; Binding constant;

LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma by Shouhong Gao; Haijun Miao; Xia Tao; Bo Jiang; Ying Xiao; Fei Cai; Yunlei Yun; Jingxian Li; Wansheng Chen (1939-1944).
A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC–MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C8 column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol-10 mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.
Keywords: Liquid chromatography-tandem mass spectroscopy; Valproic acid; Metabolites; Human plasma; Epileptic patients;

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC–MS n ) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS n . The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7–2000, 6–2000 and 8–7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.
Keywords: Ergosterol; RRLC–DAD–APCI–MS n ; RRLC–APCI–MS/MS; Pharmacokinetics; Metabolite; Elimination pathway;

Analyte and internal standard cross signal contributions and their impact on quantitation in LC–MS based bioanalysis by Aimin Tan; Isabelle A. Lévesque; Isabelle M. Lévesque; François Viel; Nadine Boudreau; Ann Lévesque (1954-1960).
Cross signal contributions between an analyte and its internal standard (IS) are very common due to impurities in reference standards and/or isotopic interferences. Despite the general awareness of this issue, how exactly they affect quantitation in LC–MS based bioanalysis has not been systematically evaluated. In this research, such evaluations were performed first by simulations and then by experiments using a typical bioanalytical method for tiagabine over the concentration range of 1–1000 ng/mL in human EDTA K3 plasma. The results demonstrate that when an analyte contributes to IS signal, linearity and accuracy can be affected with low IS concentration. Thus, minimum IS concentrations have been obtained for different combinations of concentration range, percentage of cross contribution, and weighting factor. Moreover, while impurity in analyte reference standard is a factor in cross signal contribution, significant systematic errors could exist in the results of unknown samples even though the results of calibration standards and quality controls are acceptable. How these systematic errors would affect stability evaluation, method transfer, and cross validation has also been discussed and measures to reduce their impact are proposed. On the other hand, the signal contribution from an IS to the analyte causes shifting of a calibration curve, i.e. increase of intercept, and theoretically, the accuracy is not affected. The simulation results are well supported by experimental results. For example, good inter-run (between-run) accuracy (bias: −2.70 to 5.35%) and precision (CV: 2.07–10.50%) were obtained when runs were extracted with an IS solution containing 1-fold of the lower limit of quantitation.
Keywords: Reference standard purity; Cross-contribution; Bioanalysis; Quantitation; Mass spectrometry; LC–MS; Tiagabine;

Chlorpyrifos and cypermethrin are the most used insecticides in Taiwan. Exposure to both pesticides has been associated with reproductive and developmental health effects in humans and animals. This study describes an online solid-phase extraction coupled with liquid chromatography–heated electrospray ionization tandem mass spectrometry (online SPE-LC/HESI/MS/MS) method to analyze chlorpyrifos and cypermethrin in cord blood of pregnant women. Calibration curves showed good linearity (r 2  > 0.998) for both pesticides within the range of 0.1–100 ppb. Limits of detection (LODs) were 0.01 and 0.05 ppb and recoveries in cord blood were 97.2 ± 4.8% and 93.5 ± 9.5% for chlorpyrifos and cypermethrin respectively. After analysis of 396 samples, the mean concentrations of chlorpyrifos and cypermethrin were 0.38 and 1.08 ppb respectively. These results demonstrate that LC/HESI/MS/MS is effective for the simultaneous analysis of chlorpyrifos and cypermethrin in cord blood with excellent sensitivity and specificity and may also be effective for high throughput assay in future epidemiology studies.
Keywords: Pesticide; Chlorpyrifos; Cypermethrin; Organophosphate; Pyrethroid; Cord blood; Online SPE; LC/MS/MS;

The aim of this study is to develop a simple and applicable HPLC method for the detection of vincristine in rat plasma after administration of poly(lactic-co-glycolic acid)–poly(ethylene glycol) (PLGA–PEG) nanoparticles loaded with vincristine sulfate (VCR). Vincristine was extracted from rat plasma and vinblastine sulfate was chosen as the internal standard (IS). Chromatographic separation of VCR and IS was achieved by a Dikma Dimonsil C18 column (200 mm × 4.6 mm) with the mobile phase consisting of 0.02 M sodium dihydrogen phosphate–methanol (36:64, v/v, pH = 4.7) at a flow rate of 1.0 mL/min. The ultraviolet detection wavelength was set at 276 nm. The calibration curve was linear over a concentration range of 0.05–5.0 μg/mL. The intra-day and inter-day accuracy for three quality controls (QC) samples was 93.48–107.74% and 92.61–96.58%, respectively; the precision was less than 9%. The average method recoveries for vincristine from spiked plasma at all QC levels were over 83%; and extraction recoveries were between 66 and 70%. Vincristine was stable in rat plasma for one month at −80 °C, for 8 h at room temperature, as well as during three freeze–thaw cycles. This HPLC method was applied successfully to the pharmacokinetic study of vincristine in rats after a single intravenous injection of VCR in physiological saline (F-VCR) solution, VCR-loaded PLGA–mPEG nanoparticles with (NP1) and PLGA–PEG–folate nanoparticles (NP2) suspension, respectively. There were significant differences in main pharmacokinetic parameters between F-VCR and the nanoparticles. Both kinds of VCR-loaded nanoparticles displayed improved pharmacokinetic profiles.
Keywords: Vincristine; HPLC; Pharmacokinetics; PLGA–PEG; Nanoparticle;

A rapid, sensitive and selective method was developed for the quantitative determination of hederagenin in rat plasma and cerebrospinal fluid (CSF) by ultra fast liquid chromatography–tandem mass spectrometry (UFLC–MS/MS). It has been successfully applied in a pharmacokinetic study of hederagenin in the central nervous system (CNS). Sample pretreatment involved a simple protein precipitation with methanol and a one-step extraction with ethyl acetate. Separation was carried out in a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d., 2.1 μm) column with gradient elution at a flow rate of 0.35 mL/min. The mobile phase was 5 mM ammonium acetate and acetonitrile. Detection was performed in a triple-quadruple tandem mass spectrometer by multiple-reaction-monitoring mode via electrospray ionization. A linear calibration curve for hederagenin was obtained over a concentration range of 0.406 (lower limit of quantification, LLOQ) to 203 ng/mL (r 2  > 0.99) for both plasma and CSF. The intra-day and inter-day precision (relative standard deviation, RSD) values were less than 15%. At all quality control (QC) levels, the accuracy (relative error, RE) was within −9.0% and 11.1% for plasma and CSF, respectively. The pharmacokinetics results indicated that hederagenin could pass through the blood–brain barrier. This UFLC–MS/MS method demonstrates higher sensitivity and sample throughput than previous methods. It was also successfully applied to the pharmacokinetic study of hederagenin following oral administration of Fructus akebiae extract in rats.
Keywords: Fructus akebiae; Hederagenin; UFLC–MS/MS; Pharmacokinetic; Plasma and CSF;

A simple, quick and economical liquid chromatographic/tandem mass spectrometry (LC–MS/MS) method for the quantitative determination of amoxicillin in bovine muscle was developed and validated. The sample preparation procedure involved a liquid extraction with water, followed by a protein precipitation step with acetonitrile. The extract was purified by a liquid–liquid partition with dichloromethane and the upper aqueous layer was directly injected into the LC–MS/MS system. Chromatographic separation was achieved on a reversed phase column, using a mixture of acetonitrile, water and 0.005% formic acid in water as mobile phase. Gradient elution was performed at a flow rate of 0.2 mL min−1. Amoxicillin was detected using positive electrospray ionization in selected reaction monitoring (SRM) mode and was quantified using terbutaline as internal standard. The responses for standards prepared in solvent and in matrix were equivalent and additionally the absence of signal suppression was confirmed by the post column infusion technique. Amoxicillin stability in standard solution and in matrix was investigated at different times and storage conditions. Amoxicillin standards prepared in water were stable on storage up to 20 days at −20 °C. Amoxicillin stability in matrix (spiked bovine muscle samples) was assessed up to 15 days at −20 °C. The method was validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, linearity, trueness, precision, decision limit (CCα) and detection capability (CCβ). All the trueness values fell within a range between 14.5% and 6.3%. Precision values for all levels of concentration tested were lower than the relative limit calculated by the Horwitz equation. The amoxicillin MRL is set at 50 μg kg−1 and the CCα and CCβ of the method were 61.2 μg kg−1 and 72.4 μg kg−1, respectively.
Keywords: Amoxicillin; LC–MS/MS; Bovine muscle;

Identification of hyperoside metabolites in rat using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry by Jianming Guo; Caifu Xue; Er-xin Shang; Jin-ao Duan; Yuping Tang; Dawei Qian (1987-1992).
In this paper, ultra performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF) with automated data analysis software (Metabolynx™) were applied for fast analysis of hyperoside metabolites in rat after intravenous administration. MSE was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 metabolites in rat plasma, urine and bile. The results indicated that methylation, sulfation and glucuronidation were the major metabolic pathways of hyperoside in vivo, and among them, 3′-O-methyl-hyperoside was confirmed by matching its fragmentation patterns with standard compound. The present study provided important information about the metabolism of hyperoside which will be helpful for fully understanding the mechanism of this compound's action. Furthermore, this work demonstrated the potential of the UPLC/QTOFMS approach using Metabolynx for fast and automated identification of metabolites of natural product.
Keywords: Hyperoside; UPLC/QTOFMS; Metabolynx;

A pre-column dansylated ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–MS/MS) method for simultaneous determination of risperidone (RIP), 9-hydroxyrisperidone (9-OH-RIP), monoamine and amino acid neurotransmitters in human urine was developed with the aim of providing data on how neurotransmitters may influence each other or change simultaneously in response to risperidone treatment. MultiSimplex based on the simplex algorithm and the fuzzy set theory was applied to the optimization of chromatographic separation and dansyl derivatization conditions during method development. This method exhibited excellent linearity for all the analytes with regression coefficients higher than 0.997. The lower limit of quantification (LLOQ) values for 9-OH-RIP and RIP were 0.11 and 0.06 ng/ml, respectively, and for neurotrasmitters ranged from 0.31 to 12.8 nM. The mean accuracy ranged from 94.7% to 108.5%. The mean recovery varied between 81.6% and 97.5%. All the RSD of precision and stability were below 9.7%. Finally, the optimized method was applied to analyze the first morning urine samples of schizophrenic patients treated with risperidone and healthy volunteers.
Keywords: MultiSimplex optimization; Neurotransmitter; Dansyl chloride; UPLC–MS/MS; Urine;

Simultaneous analysis of amoxicillin and sulbactam in human plasma by HPLC-DAD for assessment of bioequivalence by Qi Pei; Guo-Ping Yang; Zuo-Jun Li; Xiang-Dong Peng; Jing-Hui Fan; Zhao-Qian Liu (2000-2004).
A simple and accurate high-performance liquid chromatography with diode array detection-based (HPLC-DAD) method has been developed and validated for simultaneous determination of amoxicillin and sulbactam in human plasma. Sample preparation was involved in protein precipitation with acetonitrile followed by one-step extraction procedure. Chromatographic separation was achieved on a C18 column with an isocratic mobile phase consisting of water (containing 30 mM potassium dihydrogen phosphate, pH 2.8) and acetonitrile. The detection wavelengths of a diode array detector were set at 210 nm for amoxicillin and sulbactam, and 263 nm for the internal standard (cefadroxil). The method was validated for linearity, accuracy, precision, and stability. The calibration curve was linear from 0.163 to 14.7 μg/mL with correlation coefficient squared of 0.9991 for amoxicillin and 0.250–15.0 μg/mL with correlation coefficient squared of 0.9988 for sulbactam using 500 μL plasma samples. The lower limit of quantification was 0.163 and 0.250 μg/mL for amoxicillin and sulbactam, respectively. The imprecisions of intra- and inter-day validations for amoxicillin and sulbactam were <11% and their accuracies (%) were within the range of 95.4–105.7%. Mean recoveries were 75.9, 72.8, and 70.0% for amoxicillin, sulbactam, and cefadroxil, respectively. The established method was successfully applied to a bioequivalence study of two combination formulations of amoxicillin and sulbactam pivoxil in healthy male volunteers.
Keywords: Amoxicillin; Sulbactam; Human plasma; HPLC-DAD; Bioequivalence;

The RP-HPLC method for a simultaneous separation and quantitation of the dinitrophenyl derivative of cystathionine (N,N′-di-DNP) in biological samples together with GSH, GSSG, cysteine and cystine, provides a very useful tool for investigation of the transsulfuration pathway in biological samples, at the same providing results which reflect the redox status (GSH/GSSG ratio) and the potential of the generation of H2S. An application of the method for the study of the process of transsulfuration in various human brain regions shows the presence of cystathionine in all the investigated regions; it also demonstrates that cystathionine levels vary greatly between particular regions. The highest level in the thalamus and the lowest in the cerebellum were associated with respectively a low or high γ-cystathionase activity, and at the same time, a high cysteine and GSH level in the thalamus and a low value in the cerebellum. Based on the above results, one may suggest a regulatory mechanism responsible for inhibition of the CGL activity at high concentration values of cysteine and/or GSH. Simultaneous determinations of GSH and GSSG levels allow for determining the GSH/GSSG ratio, which reflects tissue redox status. The method may be also employed in determining the activity of γ-cystathionase and cystathionine-β synthase.
Keywords: Cystathionine; Cysteine; Glutathione; Human brain; RP-HPLC;