Journal of Chromatography B (v.983-984, #C)
Editorial Board (i).
Development and validation of determinative and confirmatory LC–MS/MS methodologies for total florfenicol and tulathromycin residues in bovine, equine and porcine kidney, liver and muscle tissues by Rick W. Fedeniuk; Del McKenzie; Massey Mizuno; Connie Neiser; Collin O’Byrne; Bryn Shurmer (1-9).
Separate methods for the quantitation and confirmation of regulatory relevant residue concentrations of total florfenicol and tulathromycin residues in multiple tissue matrices were developed and validated. Total florfenicol residues, determined and expressed as florfenicol amine (FFA) equivalents, were quantified and confirmed over a concentration range of 100–4000 ng/g, with an LOD of 33 ng/g, while total tulathromyicn residues, determined as CP-60,300 and expressed as tulathromycin equivalents, were quantified and confirmed over a concentration range of 500–10,000 ng/g, with an LOD of 300 ng/g. A 2 or 1 h acid digestion for the FFA and tulathromycin methods, respectively, followed by extraction, cleanup, and concentration using mixed-mode strong cation-exchange SPE cartridges was used. Quantitation and confirmation were accomplished using commercially available tri-deuterated FFA (FFA-D3) as internal standard and multiple reaction monitoring (MRM) of three transitions per target analyte. Mean recoveries and matrix effects were 60% and 25%; and 100% and 110%, respectively for the target analytes florfenicol amine and CP-60,300. Intra-lab method reproducibilities ranged from 7 to 11% RSD, which are within the AOAC recommended HORRATr guidelines for method reproducibilities estimated from single laboratory validation studies. Blind spikes showed that method bias was generally less than 15% for both methods within the calibration range. Both methods have been shown to meet requirements for use in national chemical residue monitoring programs.
Keywords: Acid-hydrolysis; CP-60,300; Florfenicol; Matrix effects; Tulathromycin; Variance components;
Simultaneous determination of six alkaloid components in rat plasma and its application to pharmacokinetic study of Danmu preparations by an ultra fast liquid chromatography–electrospray ionization-tandem mass spectrometry by Rong Yin; Jiaquan Chen; Yonggang Zhao; Xiaobin Jia; Zhiyuan Zhang; Liang Feng; Hui Wang; Jingjing Wang; Fenxia Zhu (10-17).
Danmu injection and Danmu tablet are two widely used traditional Chinese medicine made of Nauclea officinalis (commonly known as Danmu), in which the alkaloids are the major active substances. In this paper, an ultra fast liquid chromatography–tandem mass spectrometry (UFLC–MS/MS) method was developed for simultaneous determination and the pharmacokinetic characteristics study of six main active alkaloids (naucleamide A-10-O-β-d-glucopyranosid, naucleamide G, pumiloside, 3-epi-pumiloside, strictosamide and vincosamide) of the two above-mentioned Danmu preparations in rat plasma. In the course of the experiment, following sample preparation by protein precipitation with methanol–ethyl acetate (2:1, v/v), the nitrogen-dried extraction was reconstituted in methanol and assayed on a C18 column using a gradient elution program with mobile phase consisting of acetonitrile and water containing 0.1% formic acid. The MS detection was performed in positive ionization mode with selected ion transitions. The established method was fully validated and proved to be sensitive and specific with lower limits of quantification (LLOQs) all less than 0.32 ng/mL in rat plasma and matrix effects ranged from 88.87 to 108.27%. Good linearities of six alkaloids were obtained in respective concentration ranges (r 2 > 0.995). The average extract recoveries for each compound at three quality control concentration levels were no less than 79.70%, and the precision and accuracy were within the acceptable limits. The validated method was successfully applied to the pharmacokinetic study of six alkaloid components of Danmu injection and tablet in rat plasma. The obtained results may be helpful to reveal the action mechanism and guide the clinical application of Danmu preparations.
Keywords: UFLC–MS/MS; Danmu injection; Danmu tablet; Alkaloid components; Pharmacokinetic study;
Liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of simvastatin, lovastatin, atorvastatin, and their major metabolites in human plasma by Jiang Wang; Jasmine A. Luzum; Mitch A. Phelps; Joseph P. Kitzmiller (18-25).
Millions of individuals are treated with a variety of statins that are metabolized to a variety of active metabolites. A single assay capable of simultaneously quantifying commonly used statins and their major metabolites has not been previously reported. Herein we describe the development and validation of a novel and robust liquid chromatography–tandem mass spectrometry assay for simultaneously quantifying simvastatin, lovastatin, atorvastatin, and their metabolites, simvastatin acid, lovastatin acid, para-hydroxy atorvastatin, and ortho-hydroxy atorvastatin in human plasma. Plasma samples were processed with a simple protein precipitation technique using acetonitrile, followed by chromatographic separation using an Agilent Zorbax Extend C18 column. A 12.0 min linear gradient elution was used at a flow rate of 400 μL/min with a mobile phase of water and methanol, both modified with 2 mM ammonium formate and 0.2% formic acid. The analytes and internal standard, hesperetin, were detected using the selected reaction monitoring mode on a TSQ Quantum Discovery mass spectrometer with positive electrospray ionization. The assay exhibited a linear range of 1–1000 nM for simvastatin acid and lovastatin acid, and a linear range of 0.1–100 nM for the other analytes in human plasma. The accuracy and the within- and between-day precisions of the assay were within acceptable ranges, and the method was successfully utilized to quantify the statins and their metabolites in human plasma samples collected from an ongoing pharmacokinetic study.
Keywords: Statin; LC–MS/MS; Validation; Metabolite; Human plasma;
Preparation and characterization of novel IgG affinity resin coupling anti-Fc camelid single-domain antibodies by Zhui Tu; Yang Xu; Jinheng Fu; Zhibing Huang; Yao Wang; Bin Liu; Yong Tao (26-31).
This work aimed to evaluate novel affinity resin used to purify immunoglobulin G (IgG) with a variable domain of the heavy chain of the heavy-chain antibody (VHH) as an affinity ligand. The VHH, isolated from a naïve camelid single-domain phage display library, exhibits not only affinity to the fragment crystallizable (Fc) region of IgG but also high thermal stability. This anti-Fc VHH (AFV) was expressed as a soluble protein in Escherichia coli and purified using a simple heat treatment procedure. The effects of pH and NaCl concentrations on the capacity of AFV resin were also investigated. Results showed a robust property of the AFV resin. It could bind IgGs at various pH conditions (from 6.0 to 9.0) and NaCl concentrations. The static binding capacities of AFV resin ranged from 3.40 ± 0.53 mg/ml to 15.04 ± 0.37 mg/ml measured using rabbit, mouse, and human IgGs. The bound IgGs can be efficiently eluted at pH 5.0, which is conducive to acid-sensitive IgGs and prevents the aggregation of IgGs. After 10 purification cycles or a 7-day period of storage at 37 °C, recovery did not decrease. These findings suggested that VHHs from non-immunized library could also be robust and functional reagent as an affinity purification ligand.
Keywords: Affinity chromatography; Purification of IgG; Single domain antibody; Anti-Fc;
Rapid preparation of molecularly imprinted polymers by microwave-assisted emulsion polymerization for the extraction of florfenicol in milk by Haiyan Chen; Sunil Son; Fengshuang Zhang; Jin Yan; Yi Li; Hong Ding; Lan Ding (32-38).
In this study, we proposed a rapid and simple method for the preparation of molecularly imprinted polymers (MIPs) by emulsion polymerization. The polymerization process was accelerated by microwave heating, and the reaction time was greatly shortened. The obtained MIPs were spherical in shape and exhibited a uniform morphology. The MIPs with selectivity and high affinity to florfenicol were successfully applied as solid-phase extraction materials to extract and clean up the florfenicol in milk, followed by liquid chromatography–tandem mass spectrometry (LC–MS) analysis. The parameters affecting the performance of extraction and LC–MS analysis were evaluated. The detection limit of the method was 4.1 ng mL−1. The relative standard deviations of intra- and inter-day were in the range of 3.5–4.7% and 3.9–7.5%, respectively.
Keywords: Molecularly imprinted polymers; Microwave heating; Liquid chromatography–tandem mass spectrometry; Florfenicol; Milk;
Metabolites identification of glycyrin and glycyrol, bioactive coumarins from licorice by Qi Wang; Xue Qiao; Yi Qian; Chun-fang Liu; Yan-fang Yang; Shuai Ji; Jun Li; De-an Guo; Min Ye (39-46).
Coumarins are an important group of bioactive constituents in licorice (Glycyrrhiza uralensis), a worldwide popular herbal medicine. This study aims to elucidate the metabolism of two major licorice coumarins, glycyrin and glycyrol in rats. After oral administration of 40 mg/kg glycyrin, neither the parent compound nor its metabolites could be detected in rats plasma or urine samples, indicating that glycyrin had poor oral bioavailability. Two hydroxylated metabolites, 4″-hydroxyl glycyrin and 5″-hydroxyl glycyrin, were detected in rat liver microsome incubation system. Among them, the major metabolite 4″-hydroxyl glycyrin, which is a new compound, was obtained by microbial transformation of Syncephalastrum racemosum AS 3.264. Its structure was fully identified by 1D and 2D NMR. Meanwhile, glycyrol, together with three metabolites, were detected in rats urine and fecal samples after oral administration (40 mg/kg). Their structures were tentatively characterized by LC/MS. Glycyrol mainly undertakes hydroxylation metabolism, accompanied by hydration and dehydrogenation as minor reactions. This is the first systematic study on metabolism of glycyrin and glycyrol. The results could be valuable to evaluate druggability of these bioactive natural products.
Keywords: Coumarin; Glycyrrhiza uralensis; Glycyrin; Glycyrol; Metabolites identification;
Development of a more specific and accurate multiple reaction monitoring method based on GC–EI/MS/MS for simultaneously monitoring and determining 34 kinds of pesticides in Qianjinzhidai pills by Yunbin Jiang; Mei Zhong; Yan Gou; Wei Peng; Juan Zhou; Pie Wu; Yuying Ma (47-54).
A rapid and accurate multi-residue method was developed for simultaneously monitoring and determining 34 kinds of pesticides by GC–EI/MS/MS, successfully applied in Qianjinzhidai pills. Precision, repeatability, stability, limit of detection (LOD), limit of quantification (LOQ), linearity range and accuracy tests were used to validate this method. The results, precision (RSD < 2%, n = 6), repeatability (RSD < 2%, n = 6) and stability (RSD < 2%, n = 6), LOD (lower than 0.02 mg/kg), LOQ (lower than 0.067 mg/kg), linearity range (0.016–39.270 μg/mL), recoveries (90–110%, RSD < 15%, n = 6), indicated that the method was feasible. This paper provided an optimization method about ion transitions which can improve the specificity of mass spectrum monitoring and the accuracy of content determination. This method can be also used to monitor and determine pesticide residues of other traditional Chinese medicine, but it was worth noting that the ion transitions should been again optimized according to the sample matrix.
Keywords: GC–EI/MS/MS; Pesticide residues; Ion transitions; Monitoring; Content determination; Qianjinzhidai pills;
Simultaneous derivatization and solid-based disperser liquid–liquid microextraction for extraction and preconcentration of some antidepressants and an antiarrhythmic agent in urine and plasma samples followed by GC-FID by Mir Ali Farajzadeh; Parisa Khorram; Houshang Ghorbanpour (55-61).
The present work is based on a one-step method including derivatization and solid-based disperser liquid–liquid microextraction followed by gas chromatography-flame ionization detection (GC-FID) for the determination of four antidepressants (fluoxetine, fluvoxamine, tranylcypromine, and nortriptyline) and an antiarrhythmic agent (mexiletine) in human urine and plasma samples. In this method, a mixture of 1,1,2,2-tetrachloroethane (extraction solvent) and butylchloroformate (derivatizing reagent) is added on a sugar cube (solid disperser) and it is introduced into an aqueous sample containing the analytes and a catalyst, e.g. 3-methylpyridine (picoline). During dissolving the sugar cube by manual shaking, the extractant and derivatization agent are gradually released into the sample as very fine droplets. Then the resulted cloudy solution is centrifuged and the sedimented phase is analyzed by GC-FID. The influence of several variables on the efficiency of derivatization/microextraction procedure such as kind and volume of extraction solvent, type and amount of disperser, amount of derivatization agent, and catalyst volume are optimized. Under the optimum conditions the calibration curves are linear in the range of 8–100,000 μg L−1 (coefficient of determination ≥0.994). The relative standard deviations are obtained in the range of 3.0–6.0% for all compounds. Moreover, the detection limits and enrichment factors of the target analytes are obtained in the ranges 1–15 μg L−1 and 228–268, respectively, for both plasma and urine samples. The relative recoveries obtained for the spiked plasma and urine samples are between 70 and 91%. The results show that the proposed method is simple, reliable, low cost, and applicable to determine trace amounts of the studied drugs in biological samples.
Keywords: Antidepressants; Biological samples; Derivatization; Gas chromatography; Microextraction; Solid disperser;
Development and validation of an LC-MS/MS method for the determination of pelubiprofen and its active metabolite, trans-alcohol, in human plasma and its application to pharmacokinetic study by Ju-Hee Ryu; Ji-Sun Park; Min-ho Jo; Joo-Il Kim; Wang-Seob Shim; Bo-Hyung Kim; Sung-Vin Yim; Jongki Hong; Kyung-Tae Lee (62-67).
A suitable liquid chromatography tandem mass spectrometry (LC-MS/MS) method is required to determine pelubiprofen and its active metabolite, trans-alcohol (M-D), in human plasma for pharmacokinetic studies of pelubiprofen preparations. After one-step liquid–liquid extraction (LLE) using methyl tert-butyl ether (MTBE), pelubiprofen, M-D, and tolbutamide (the internal standard, IS) were eluted from a Capcellpak C18 ACR column using a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile at a flow rate 0.35 mL/min. The achieved lower limits of quantitation (LLOQ) of pelubiprofen and M-D were both 15 ng/mL (S/N > 10) and the standard calibration curves for pelubiprofen and M-D were linear (correlation coefficients >0.99) over the studied concentration range (15–2000 ng/mL). Intra- and inter-day precisions were within 7.62% for all analytes and the deviation of assay accuracies was within ±13.23%. The developed method was successfully applied to a pharmacokinetic study of pelubiprofen in healthy Korean male volunteers.
Keywords: Pelubiprofen; Metabolite; LC-MS/MS; Liquid–liquid extraction; Human plasma; Pharmacokinetic study;
Large-scale retrospective evaluation of regulated liquid chromatography–mass spectrometry bioanalysis projects using different total error approaches by Aimin Tan; Taoufiq Saffaj; Adrien Musuku; Kayode Awaiye; Bouchaib Ihssane; Fayçal. Jhilal; Saad. Alaoui Sosse; Fethi Trabelsi (68-75).
The current approach in regulated LC–MS bioanalysis, which evaluates the precision and trueness of an assay separately, has long been criticized for inadequate balancing of lab-customer risks. Accordingly, different total error approaches have been proposed. The aims of this research were to evaluate the aforementioned risks in reality and the difference among four common total error approaches (β-expectation, β-content, uncertainty, and risk profile) through retrospective analysis of regulated LC–MS projects. Twenty-eight projects (14 validations and 14 productions) were randomly selected from two GLP bioanalytical laboratories, which represent a wide variety of assays. The results show that the risk of accepting unacceptable batches did exist with the current approach (9% and 4% of the evaluated QC levels failed for validation and production, respectively). The fact that the risk was not wide-spread was only because the precision and bias of modern LC–MS assays are usually much better than the minimum regulatory requirements. Despite minor differences in magnitude, very similar accuracy profiles and/or conclusions were obtained from the four different total error approaches. High correlation was even observed in the width of bias intervals. For example, the mean width of SFSTP's β-expectation is 1.10-fold (CV = 7.6%) of that of Saffaj–Ihssane's uncertainty approach, while the latter is 1.13-fold (CV = 6.0%) of that of Hoffman–Kringle's β-content approach. To conclude, the risk of accepting unacceptable batches was real with the current approach, suggesting that total error approaches should be used instead. Moreover, any of the four total error approaches may be used because of their overall similarity. Lastly, the difficulties/obstacles associated with the application of total error approaches in routine analysis and their desirable future improvements are discussed.
Keywords: Total error; Regulated bioanalysis; LC–MS; Retrospective analysis; Uncertainty;
A validated UPLC–MS/MS assay using negative ionization mode for high-throughput determination of pomalidomide in rat plasma by Muzaffar Iqbal; Essam Ezzeldin; Khalid A. Al-Rashood; Faiyaz Shakeel (76-82).
In this study, a sensitive UPLC–MS/MS assay was developed and validated for high-throughput determination of pomalidomide in rat plasma using celecoxib as an internal standard (IS). Liquid liquid extraction using dichloromethane was employed to extract pomalidomide and IS from 200 μL of plasma. Chromatographic separation was carried on Acquity BEH™ C18 column (50 mm × 2.1 mm, 1.7 μm) using an isocratic mobile phase of acetonitrile: 10 mM ammonium acetate (80:20, v/v), at a flow rate of 0.250 mL/min. Both pomalidomide and IS were eluted at 0.66 ± 0.03 and 0.80 ± 0.03 min, respectively, with a total run time of 1.5 min only. A triple quadruple tandem mass spectrometer using electrospray ionization in negative mode was employed for analyte detection. The precursor to product ion transitions of m/z 272.01 → 160.89 for pomalidomide and m/z 380.08 → 316.01 for IS were used to quantify them respectively, multiple reaction monitoring mode. The developed method was validated according to regulatory guideline for bioanalytical method validation. The linearity in plasma sample was achieved in the concentration range of 0.47–400 ng/mL (r 2 ≥ 0.997). The intra and inter-day precision values were ≤11.1% (RSD, %) whereas accuracy values ranged from −6.8 to 8.5% (RE, %). In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of pomalidomide in rats.
Keywords: Pomalidomide; UPLC–MS/MS; Pharmacokinetics; Rat plasma;
Elimination of chromatographic and mass spectrometric problems in GC–MS analysis of Lavender essential oil by multivariate curve resolution techniques: Improving the peak purity assessment by variable size moving window-evolving factor analysis by Mehdi Jalali-Heravi; Roudabeh Sadat Moazeni-Pourasil; Hassan Sereshti (83-89).
In analysis of complex natural matrices by gas chromatography–mass spectrometry (GC–MS), many disturbing factors such as baseline drift, spectral background, homoscedastic and heteroscedastic noise, peak shape deformation (non-Gaussian peaks), low S/N ratio and co-elution (overlapped and/or embedded peaks) lead the researchers to handle them to serve time, money and experimental efforts. This study aimed to improve the GC–MS analysis of complex natural matrices utilizing multivariate curve resolution (MCR) methods. In addition, to assess the peak purity of the two-dimensional data, a method called variable size moving window-evolving factor analysis (VSMW-EFA) is introduced and examined. The proposed methodology was applied to the GC–MS analysis of Iranian Lavender essential oil, which resulted in extending the number of identified constituents from 56 to 143 components. It was found that the most abundant constituents of the Iranian Lavender essential oil are α-pinene (16.51%), camphor (10.20%), 1,8-cineole (9.50%), bornyl acetate (8.11%) and camphene (6.50%). This indicates that the Iranian type Lavender contains a relatively high percentage of α-pinene. Comparison of different types of Lavender essential oils showed the composition similarity between Iranian and Italian (Sardinia Island) Lavenders.
Keywords: Lavender; Essential oil; GC–MS; MCR;
Validation of an analytical method for nitrous oxide (N2O) laughing gas by headspace gas chromatography coupled to mass spectrometry (HS-GC–MS): Forensic application to a lethal intoxication by N. Giuliani; J. Beyer; M. Augsburger; V. Varlet (90-93).
Drug abuse is a widespread problem affecting both teenagers and adults. Nitrous oxide is becoming increasingly popular as an inhalation drug, causing harmful neurological and hematological effects. Some gas chromatography–mass spectrometry (GC–MS) methods for nitrous oxide measurement have been previously described. The main drawbacks of these methods include a lack of sensitivity for forensic applications; including an inability to quantitatively determine the concentration of gas present. The following study provides a validated method using HS-GC–MS which incorporates hydrogen sulfide as a suitable internal standard allowing the quantification of nitrous oxide. Upon analysis, sample and internal standard have similar retention times and are eluted quickly from the molecular sieve 5 Å PLOT capillary column and the Porabond Q column therefore providing rapid data collection whilst preserving well defined peaks. After validation, the method has been applied to a real case of N2O intoxication indicating concentrations in a mono-intoxication.
Keywords: Nitrous oxide; Inhalation drug abuse; Gas analysis; HS-GC–MS;
Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel by M. Ashraf-Khorassani; J. Yang; P. Rainville; M.D. Jones; K.J. Fountain; G. Isaac; L.T. Taylor (94-100).
Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2 μm particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0 mm × 150 mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10 min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an “in-house” synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica.
Keywords: Ultrahigh performance supercritical fluid chromatography; Tobacco seed oil; Normal phase chromatography; Evaporative light scattering detection; Biodiesel purity test; Octa-decyl bonded silica particles;
Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies by Bernd Moritz; Volker Schnaible; Steffen Kiessig; Andrea Heyne; Markus Wild; Christof Finkler; Stefan Christians; Kerstin Mueller; Li Zhang; Kenji Furuya; Marc Hassel; Melissa Hamm; Richard Rustandi; Yan He; Oscar Salas Solano; Colin Whitmore; Sung Ae Park; Dietmar Hansen; Marcia Santos; Mark Lies (101-110).
Within pharmaceutical industry charge heterogeneity testing of biopharmaceuticals has to be reproducible and fast. It should pass method validation according to ICH Q2. Classical approaches for the analysis of the charge heterogeneity of biopharmaceuticals are ion exchange chromatography (IEC) and isoelectric focusing (IEF). As an alternative approach, also capillary zone electrophoresis (CZE) was expected to allow reliable charge heterogeneity profiling by separation according to the analyte's net charge and hydrodynamic radius.Aim of this study was to assess if CZE possesses all of the required features. Therefore, beside lab internal validation of this method also an international cross company study was organized.It was shown that CZE is applicable across a broad pI range between 7.4 and 9.5. The coefficient of correlation was above 0.99 which demonstrated linearity. Precision by repeatability was around 1% (maximum relative standard deviation per level) and accuracy by recovery was around 100% (mean recovery per level). Accuracy was further verified by direct comparison of IEC, IEF and CZE, which in this case showed comparable %CPA results for all three methods. However, best resolution for the investigated MAb was obtained with CZE. In dependence on sample concentration the detection limit was between 1 and 3%.Within the intercompany study for CZE the same stressed and non-stressed samples were analyzed in each of the 11 participating labs. The finally obtained dataset contained more than 1000 separations which provided an extended dataset for further statistical evaluation. Among the different labs no significant differences between the peak profiles were observed. Mean driver for dropouts in quantitative evaluation was linked to the performance of some participating labs while the impact of the method performance was negligible. In comparison to a 50 cm capillary there was a slightly better separation of impurities and drug substance related compounds with a 30 cm capillary which demonstrates that an increased stability indicating potential can be combined with the increased separation velocity and high throughput capability of a shorter capillary. Separation can be performed in as little as approx. 3 min allowing high throughput applications. The intercompany study delivered precise results without explicit training of the participating labs in the method prior to the study (standard deviations in the range of 1%).It was demonstrated that CZE is an alternative platform technology for the charge heterogeneity testing of antibodies in the pharmaceutical industry.
Keywords: Capillary zone electrophoresis; Pharmaceuticals; Intercompany study; Antibody; Reproducibility; High throughput;
Development and validation of a high-performance liquid chromatography method for the quantification of ursolic/oleanic acids mixture isolated from Plumeria obtusa by Helen L. Alvarado; Guadalupe Abrego; María L. Garduño-Ramirez; Beatriz Clares; María L. García; Ana C. Calpena (111-116).
Oleanolic acid (OA) and ursolic acid (UA) are ubiquitous pentacyclic triterpenes compounds in plants. These triterpenoids exhibit unique, important biological and pharmacological activities. For the investigation and development of topical drug delivery systems of triterpenoids in Plumeria obtusa is essential an adequate detection and quantification method for its application in skin permeation studies. The aim of this study was to develop a HPLC method for the determination of OA/UA from leaves of P. obtusa. Results showed that it was sensitive, repeatable, selective, accurate and precise. The detection limit was 0.93 ± 0.21 μg/mL and the quantification limit 2.81 ± 0.65 μg/mL. Determination coefficients were higher than 0.999 for concentrations between 3.62 and 116 μg/mL. The intra and inter-day precision (relative standard deviation) was less than 1.50% and accuracy in terms of relative error ranged between −1.45 and 1.39%. The proposed HPLC method presented advantageous performance characteristics and it can be considered suitable for the evaluation of OA/UA in ex vivo permeation studies.
Keywords: Oleanolic acid; Ursolic acid; HPLC; Validation; Skin permeation studies; Nanoparticles;
LC–MS/MS bioanalysis of loratadine (Claritin) in dried blood spot (DBS) samples collected by subjects in a clinical research study by Wenkui Li; John Doherty; Paul Moench; Jimmy Flarakos; Francis L.S. Tse (117-124).
A high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method has been developed and validated for the quantitative analysis of loratadine, an H1 histamine antagonist, in human dried blood spot (DBS) samples following a single self-administered 10 or 20 mg oral dose. The samples were produced by spotting approximately 30 μl of whole blood onto PE-226 cards. Two 3-mm discs were cut from the DBS samples and extracted using aqueous methanol containing the internal standard. After transfer and drying of the resulting sample extract, the reconstituted residues were chromatographed using a Waters XSelect C18 column and isocratic elution for MS/MS detection. The possible impact due to hematocrit, volume of blood sample spotted, storage temperature, and humidity, on the accuracy of measured DBS results were investigated. The results showed that only spotted blood volume might have an impact; a small volume (10 μl) tended to give a larger negative bias in the measured value than the large volume ones (≥20 μl). The current method was fully validated over a dynamic range of 0.200–20.0 ng/ml with correlation coefficients (r 2) for three validation batches equal to or better than 0.990. The intra-day accuracy and precision at the LLOQ were −11.5 to 0.0% bias and 6.4 to 8.9% CV, respectively. For the other QC samples (0.600, 3.00, 10.0 and 15.0 ng/ml), the precision ranged from 4.2 to 9.8% CV and from 6.3 to 8.1% CV, respectively, in the intra-day and inter-day evaluations; the accuracy ranged from -1.7 to 10.0% and 2.7 to 5.3% bias, respectively, in the intra-day and inter-day batches. Loratadine is stable in the DBS samples for at least 271 days at ambient temperature in a desiccator, for at least 24 h at 60 °C and under 80% relative humidity, followed by re-conditioning at ambient temperature in a desiccator. The current methodology has been applied to determine the loratadine levels in DBS samples collected by subjects in a clinical research study to evaluate pharmacokinetic sampling in point-of-care setting.
Keywords: Dried blood spot (DBS); Loratadine; Blood volume; Hematocrit impact; Stability; Humidity impact; Self-sampling; Point-of-care setting; LC–MS/MS;
Pharmacokinetics and tissue distribution model of cabozantinib in rat determined by UPLC–MS/MS by Xianqin Wang; Shuanghu Wang; Feiyan Lin; Qingwei Zhang; HuiLing Chen; Xianchuan Wang; Congcong Wen; Jianshe Ma; Lufeng Hu (125-131).
Cabozantinib (XL184) is a novel small molecule inhibitor of receptor tyrosine kinases (RTKs) targeted at mesenchymal–epithelial transition factor (MET). In order to study the pharmacokinetics and tissue distribution in rat, a specific ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed with midazolam as internal standard. The calibration curves in plasma and tissues were linear in the range of 5–5000 ng/mL (r 2 > 0.99). The recoveries were better than 80.4% and matrix effects ranged from 96.9% to 105.1%. Then, the developed UPLC–MS/MS method was applied to determine the concentration of XL184 in blood and tissues. The pharmacokinetics of four different dosages (iv 5, 10 mg/kg and ig 15, 30 mg/kg) revealed that XL184 was eliminated slowly, the t 1/2 was longer than 10 h and the absolute bioavailability was 25.6 ± 8.3%. The concentration distribution of XL184 in tissues was liver > lung > kidney > spleen > heart. Based on the concentration–time of XL184 in tissues, a BP-ANN distribution model was developed with good performance, and can be used to predict the concentration of XL184 in tissues.
Keywords: Cabozantinib; UPLC–MS/MS; Pharmacokinetics; BP-ANN;