Journal of Pharmaceutical and Biomedical Analysis (v.52, #2)

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile–0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1–50 μg/ml (R 2  = 0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17 ± 0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 μg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release.
Keywords: Dapivirine; HIV-1; Antiretroviral; Microbicides; RP-HPLC; Poly(lactic-co-glycolic acid); Nanoparticles;

Quantitative determination of flavonoids and cycloartanol glycosides from aerial parts of Sutherlandia frutescens (L.) R. BR. by using LC-UV/ELSD methods and confirmation by using LC–MS method by Bharathi Avula; Yan-Hong Wang; Troy J. Smillie; Xiang Fu; Xing Cong Li; Wilfred Mabusela; James Syce; Quinton Johnson; William Folk; Ikhlas A. Khan (173-180).
This paper describes the first analytical method for the determination of four flavonoids (sutherlandins A–D) and four cycloartanol glycosides (sutherlandiosides A–D) from the aerial parts of Sutherlandia frutescens (L.) R. Br. A separation by HPLC was achieved by using a reversed phase (RP-18) column, PDA with ELS detection, and a water/acetonitrile gradient as the mobile phase. The wavelength used for quantification of four flavonoids with the diode array detector was 260 nm. Owing to their low UV absorption, the cycloartanol glycosides were detected by evaporative light scattering. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). The limits of detection and limits of quantification of eight compounds were found to be in the range from 0.1 to 7.5 μg/mL and 0.5 to 25 μg/mL, respectively. The analysis of products showed considerable variation of 1.099–5.224 mg/average weight for the major compound, sutherlandioside B. The eight compounds in plant sample and products of S. frutescens were further confirmed by LC–ESI-TOF. This method involved the use of the [M+H]+ and [M+Na]+ ions in the positive ion mode with extractive ion monitoring (EIM).
Keywords: Sutherlandia frutescens (L.) R. Br.; LC-UV–ELSD; LC–MS; Dietary supplements;

An optical method for continuous monitoring of the dissolution rate of pharmaceutical powders by Riikka Laitinen; Jani Lahtinen; Pertti Silfsten; Erik Vartiainen; Pekka Jarho; Jarkko Ketolainen (181-189).
Monitoring systems providing fast and reliable, even on-line data, from a distinct process stage or final product are needed in drug development, from the early stages of drug discovery until the drug product manufacturing procedures. This includes also processes involving solid particles, such as drug dissolution. However, the existing in vitro drug dissolution test methods suffer limitations, such as long sampling times of 30–60 s and thus the inability to be adapted to continuous monitoring, time consuming sample preparation and consumption of large amounts of reagents. In this study, an optical method for monitoring the dissolution rate of pharmaceutical powders was evaluated with model drugs having different dissolution rates. The measuring system consisted of a laser source, light detector, oscilloscope, magnetic stirrer and sample vessel. The intensity of laser light transmitted through the dissolution medium was recorded and displayed by the oscilloscope. Dissolution curves were produced by fitting the raw data with mathematical functions. The optical method was found to be resource-saving, reliable and capable of detecting differences in even rapid dissolution rates of drug compounds. This technique might have targets of application in real-time monitoring of processes in many different sectors, including the pharmaceutical industry.
Keywords: Light scattering; Dissolution rate; Dissolution monitoring; Process analytical technology;

Photohuperzine A—A new photoisomer of huperzine A: Structure elucidation, formation kinetics and activity assessment by Lygia Azevedo Marques; Martin Giera; Frans J.J. de Kanter; Wilfried M.A. Niessen; Henk Lingeman; Hubertus Irth (190-194).
A new photoisomer of the promising “anti-Alzheimer” drug candidate (±) huperzine A is described. The new substance was formed via a photoisomerization reaction and was found to be 1-amino-13-ethylidene-11-methyl-6-aza-tetracyclo-[]-trideca-2,10-diene-5-one using NMR analysis. The kinetics of its formation was studied and proven to be of first-order. The described photoisomer showed a significant loss in activity, being more than 100 times less active than (−) huperzine A itself. The new substance was named photohuperzine A, referring to its photopyridone substructure.
Keywords: Huperzine A; Photoisomerization; Kinetics; Stability; NMR;

Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH by Ruedeeporn Tantipolphan; Stefan Romeijn; John den Engelsman; Riccardo Torosantucci; Tue Rasmussen; Wim Jiskoot (195-202).
The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a “physiological” (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in “physiological” HP-SEC is affected by both dynamic association–dissociation of insulin molecules and insulin–column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from “physiological” and compendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin.
Keywords: Insulin; High-performance size exclusion chromatography (HP-SEC); Association state; Arginine; Zinc ions;

The frequently used sympathomimetic drug phenylephrine has been studied by electrospray ionisation-mass spectrometry. The stability of the adrenoceptor agonist was examined by investigations of the pharmaceutically used salts phenylephrine hydrochloride and phenylephrine bitartrate. Photostability has been studied by use of an irradiation equipment emitting a solar radiation spectrum. The experiments were carried out by analysis of aqueous drug solutions before and after irradiation treatment. The phenylephrine derivative with unsaturated side chain originating from the drug by loss of one water molecule has been detected as the major degradation product of both phenylephrine salts the hydrochloride and the bitartrate. Further degradation and oxidation products were detectable already in the full scan mode demonstrating a low stability of the drug. Tandem mass spectrometry and multiple stage mass spectrometry experiments enabled the establishment of fragmentation schemes of both salts for the first time. Irradiation treatment indicated that phenylephrine bitartrate is more prone to degradation than the hydrochloride because of an additional decomposition sensitivity of the tartaric acid counter ion. An interaction between phenylephrine and its counter ion degradation products via a nucleophilic addition mechanism is suggested to be the explanation for the detected ion signals after irradiation treatment of phenylephrine bitartrate.
Keywords: Phenylephrine; Stability; Degradation; Electrospray ionisation-mass spectrometry; Fragmentation;

The degradation of salbutamol in ethanolic solutions by Mike Cope; Fernando Bautista-Parra (210-215).
The degradation pathways of salbutamol in ethanolic solutions have been investigated and three potential ethyl ether degradation products have been identified. Two have been confirmed as salbutamol ethyl ethers and the third as a diethyl ether. All three degradation products have been structurally elucidated by LC–MS–MS (TOF and tandem quadrupole). The two ethyl ethers have a molecular weight of 267 Da (28 units higher than salbutamol) and are structural isomers (molecules with the same molecular weight but different structural arrangements). The molecular weight of the two ethyl ethers is consistent with the addition of one ethyl group to the salbutamol molecule and elimination of one water molecule. The molecular weight of the diethyl ether is 295 Da (56 units higher than salbutamol) and is consistent with the addition of two ethyl groups to the salbutamol molecule and elimination of two water molecules. A plausible degradation mechanism for the formation of the salbutamol ethyl ethers is the acid-catalysed dehydration of alcohols. Acidic pH is required to drive the degradation of salbutamol in ethanolic solution. Higher degradation levels of salbutamol ethyl ethers are achieved in acidic pH solutions. Levels of the two salbutamol ethyl ethers reach a maximum at an ethanol concentration of around 20%. Levels of the diethyl ether increase linearly with ethanol concentration, until it becomes the major degradation product in high concentration ethanolic solutions (≥30%).
Keywords: Salbutamol; Albuterol; Ethanol; Degradation; Stability; Salbutamol ethyl ethers;

Determination of gemifloxacin in different tissues of rat after oral dosing of gemifloxacin mesylate by LC–MS/MS and its application in drug tissue distribution study by Bikash Roy; Ayan Das; Uttam Bhaumik; Amlan Kanti Sarkar; Anirbandeep Bose; Jayanti Mukharjee; Uday Sankar Chakrabarty; Anjan Kumar Das; Tapan Kumar Pal (216-226).
A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to evaluate the accumulation of gemifloxacin in different tissues of Wister albino rat. The analytical method consists of the homogenization of tissues followed by simple liquid–liquid extraction and determination of gemifloxacin by an LC–MS/MS. The analyte was separated on a Peerless basic C18 column (33 mm × 4.6 mm, 3 μm) with an isocratic mobile phase of methanol–water containing formic acid (1.0%, v/v) (9:1, v/v) at a flow rate of 0.6 ml/min. The MS/MS detection was carried out by monitoring the fragmentation of m/z 390.100 → 372.100 for gemifloxacin and m/z 332.100 → 314.200 for ciprofloxacin (internal standard; IS) on a triple quadrupole mass spectrometer. The validated method was accurate, precise and rugged with good linearity in all tissue homogenates. The accuracy and precision value obtained from six different sets of quality control samples of all tissues and serum analyzed in separate occasions within 91.833–102.283% and 0.897–5.291%, respectively. The method has been successfully applied to tissue distribution studies of gemifloxacin. The present study demonstrates that the highest tissue concentration of gemifloxacin was obtained in lung (11.891 ng/g), followed by liver (10.110 ng/g), kidney (10.095 ng/g), heart (4.251 ng/g), testis (3.750 ng/g), stomach (3.182 ng/g), adipose tissue (1.116 ng/g) and brain (0.982 ng/ml) in 3 h after multiple oral dosing of 200 mg gemifloxacin mesylate for 7 days. This method may also be used for gemifloxacin tissue distribution modeling study in rat tissues and antibiotic residue analyses in other animal tissues.
Keywords: Gemifloxacin; LC–MS/MS; Quantification; Validation; Tissue distribution;

A chiral liquid chromatography–tandem mass spectrometry (LC–MS/MS) simultaneous stereoselective analysis of bambuterol and its active metabolite terbutaline enantiomers in Wistar rat plasma has been developed and validated. All analytes and the internal standard were extracted from rat plasma samples by liquid–liquid extraction, separated on macrocyclic glycopeptide teicoplanin column with mobile phase constituted of 20 mM ammonium acetate solution–methanol (10:90, v/v) at a flow-rate of 0.4 mL/min. Detection was performed on an API 3000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves in the range 1–800 ng/mL were linear and the accuracy for each analyte was within 8.0%. The intra- and inter-day precision as determined from quality control samples was less than 10.1%. The validated assay was successfully used to determine the enantiomers of bambuterol and terbutaline in rat plasma samples in the pharmacokinetic studies of rac-bambuterol.
Keywords: Stereoselective separation; Bambuterol; Terbutaline; LC–MS/MS; Plasma;

Precision in affinity capillary electrophoresis for drug–protein binding studies by Deia El-Hady; Sascha Kühne; Nagwa El-Maali; Hermann Wätzig (232-241).
In order to achieve excellent precision in the estimation of binding constants by affinity capillary electrophoresis (ACE), electroosmotic flow (EOF) stability is the key parameter, especially when using proteins in binding assays. Appropriate rinsing protocols are mandatory. In our study, the capillary was rinsed after each run with 0.1 mol/L sodium hydroxide for 2.0 min, with water for 2.0 min followed by running electrolyte (phosphate buffer at pH 7.4) for 3.0 min (pressure = 3000 mbar each). Tryptophan-human serum albumin, warfarin-bovine serum albumin and quercetin-β-lactoglobulin were used as ACE models. Further improvements in precision have been obtained by avoiding a complete standstill of liquid within the capillary and flushing the capillary with buffer for 25 min after each 30 consecutive runs. The precision of measurements is further improved by the use of mobility ratios to report mobility changes (RSD% less than 0.5% in a long-term measurement, n  = 300–600). Apart from the importance of a stable EOF, other ACE key parameters include protein concentration, drug plug length, applied voltage, and the choice of the regression method. In the present work, useful protocols and templates are provided in order to allow users a quick and efficient start with ACE methods. The comprehensive experimental part can serve as a checklist, which parameters need to be addressed for successfully applying ACE. Here, the suggested experimental design allows for the determination of binding constants within a couple of hours using standard instrumentation. This time could still be decreased by orders of magnitude using capillary arrays or miniaturized systems.
Keywords: ACE; Protein; Precision; Binding constant;

A fast and sensitive method to quantify fasudil hydrochloride (FH) and its active metabolite hydroxyfasudil (M3) in human plasma using HPLC–MS/MS has been developed and validated in present study. The method involved simple sample preparation with methanol as protein precipitation (3:1, v/v) and ranitidine as an internal standard (IS). The analytes and IS were separated using a gradient elution procedure on the analytical column ZORBAX StableBond-C18 (5 μm, 150 mm × 4.6 mm). Detection was performed by an AB 3200 QTRAP tandem mass spectrometer equipped with a Turbo IonSpray ionization source set in positive ion mode. Multiple reaction monitoring (MRM) using the precursor to product ion was m/z 292.2/99.2 for fasudil, m/z 308.2/99.2 for M3 and m/z for 315.3/176.2 for IS. The linear range of the method was from 0.4 to 250 ng/mL for both fasudil and M3. The lower limit of quantification was 0.4 ng/mL for both fasudil and M3. The intra- and inter-day relative standard deviation over the entire concentration range was less than 7.11% for fasudil and 10.6% for M3, respectively. The validated method was successfully applied for the evaluation of pharmacokinetic of fasudil hydrochloride after administration of 30 mg fasudil hydrochloride by continuous intravenous infusion over 30 min in 12 healthy Chinese volunteers.
Keywords: Fasudil; Hydroxyfasudil; HPLC–MS/MS; Validation; Pharmacokinetic;

Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development.
Keywords: Anti-drug antibody; Immune complex; Drug interference; Cynomolgus monkey; Immunogenicity;

Surface plasmon resonance based immunosensor for serological diagnosis of dengue virus infection by Sunita Kumbhat; Kavita Sharma; Rakhee Gehlot; Aruna Solanki; Vinod Joshi (255-259).
Surface plasmon resonance (SPR) is a promising tool in sensor technology for biomedical applications. An SPR based immunosensor was established for label free and real time assay for the serological diagnosis of dengue virus infection employing the dengue virus antigen as the sensing element. The dengue virus antigen conjugated with bovine serum albumin is covalently immobilized on a gold sensor chip via activated self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid, by amide coupling. Surface morphology of the biosensor was recorded using atomic force microscopy. Presence of dengue virus specific IgM antibodies in dengue positive sera was monitored by increase in resonance angle in direct immunoassay, whereas the principle of indirect competitive inhibition immunoassay was used to detect presence of dengue virus for early detection of the onset of dengue viral infection in clinical diagnostics. Results were compared with those obtained by MAC-ELISA. The regeneration was achieved by pepsin solution in glycin–HCl buffer (pH 2.2) and sensor surface displayed a high level of stability during repeated immunoreaction cycles. The proposed biosensor being simple, effective and based on utilization of natural antigen–antibody affinity, our study presents an encouraging scope for development of biosensors for diagnosis of dengue and dengue hemorrhagic fever (DHF) which continues to be a major health problem in the tropical and subtropical regions of world.
Keywords: Surface plasmon resonance; Immunosensor; Self-assembled monolayer; Serological diagnosis; Dengue;

Measurement of ethyl methanesulfonate in human plasma and breast milk samples using high-performance liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry by M. Angela Montesano; Ralph D. Whitehead; Nayana K. Jayatilaka; Peter Kuklenyik; Mark D. Davis; Larry L. Needham; Dana Boyd Barr (260-264).
Ethyl methanesulfonate (EMS) is a mesylate ester, which is known to be a potent mutagen, teratogen, and possibly carcinogen. Mesylate esters have been found in pharmaceuticals as contaminants formed during the manufacturing process and may potentially pose an exposure hazard to humans. We have developed and validated a method for detection of trace amounts (ng/ml levels) of EMS in human plasma and breast milk. The samples were extracted by matrix solid-phase dispersion with ethyl acetate using Hydromatrix™ and the ASE 200 Accelerated Solvent Extractor. The extracts were separated by high-performance liquid chromatography (HPLC) using a HILIC column. The detection was performed with a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Electron Corporation) using atmospheric pressure chemical ionization in negative-ion mode and multiple reaction monitoring. The use of a surrogate internal standard in combination with HPLC–MS/MS provided a high degree of accuracy and precision. The extraction efficiency was greater than 70%. Repeated analyses of plasma and breast milk samples spiked with high (100 ng/ml), medium (50 ng/ml) and low (5 ng/ml) concentrations of the analytes gave relative standard deviations of less than 12%. The limits of detection were in the range of 0.5–0.9 ng/ml for both matrices.
Keywords: Ethyl methanesulfonate; EMS; HPLC; Mass spectrometry; Electrospray ionization;

Plasma metabolic fingerprinting of childhood obesity by GC/MS in conjunction with multivariate statistical analysis by Maomao Zeng; Yizeng Liang; Hongdong Li; Mei Wang; Bing Wang; Xian Chen; Neng Zhou; Dongsheng Cao; Jing Wu (265-272).
Metabolic fingerprinting is a powerful tool for exploring systemic metabolic perturbations and potential biomarkers, thus may shed light on the pathophysiological mechanism of diseases. In this work, a new strategy of metabolic fingerprinting was proposed to exploit the disturbances of metabolic patterns and biomarker candidates of childhood obesity. Plasma samples from children with normal weight, overweight and obesity were first profiled by GC/MS. ULDA (uncorrelated linear discriminant analysis) then revealed that the metabolic patterns of the three groups were different. Furthermore, several metabolites, say isoleucine, glyceric acid, serine, 2,3,4-trihydroxybutyric acid and phenylalanine were screened as potential biomarkers of childhood obesity by both ULDA and CCA (canonical correlation analysis). CCA also shows satisfactory correlation between the metabolic patterns and clinical parameters, and the results further suggest that WHR (waist–hip ratio) together with TG (total triglycerides), TC (total cholesterol), HDL (high density lipoprotein) and LDL (low density lipoprotein) were the most important parameters which are associated closely with the metabolic perturbations of childhood obesity, so as to be paid more attention for dealing with metabolic disturbances of childhood obesity in clinical practice rather than regularly monitored BMI (body-mass index). The results have demonstrated that the proposed metabolic fingerprinting approach may be a useful tool for discovering metabolic abnormalities and possible biomarkers for childhood obesity.
Keywords: Canonical correlation analysis; Childhood obesity; Gas chromatography/mass spectrometry; Metabolic fingerprinting; Uncorrelated linear discriminant analysis;

Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry by Xin Hua; Yu-Jie Fu; Yuan-Gang Zu; Nan Wu; Yu Kong; Ji Li; Xiao Peng; Thomas Efferth (273-279).
Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC–MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C18 column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R  = 0.9967) for plasma samples and 10 to 6000 ng/mL (R  ≥ 0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5–104.6% and 93.3–107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T max, 10.7 ± 0.31 min; t 1/2, 51.40 ± 6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.
Keywords: Cajanus cajan; Cajaninstilbene acid (CSA); LC–MS/MS; Pharmacokinetics; Tissue distribution;

To investigate the interaction between Ractopamine (RAC), an animal growth promoter, and bovine serum albumin (BSA), three spectroscopic approaches (fluorescence, UV–vis and FT-IR) and three different experiments (two mole-ratio and a Job's methods) were used to monitor the biological kinetic interaction procedure. The Stern–Volmer quenching constants K SV, the binding constants K a, and the number of binding sites n at 298, 301 and 304 K were evaluated by molecular spectroscopic approaches. The values of enthalpy (−13.47 kJ mol−1) and entropy (78.39 J mol−1  K−1) in the reaction indicated that RAC bound to BSA mainly by electrostatic and hydrophobic interaction. The site markers competitive experiments indicated that the binding of RAC to BSA primarily took place in site I. The spectra data matrix was further investigated with multivariate curve resolution-alternating least squares (MCR-ALS), and the concentration profiles and the pure spectra for three species (BSA, RAC and RAC-BSA) existed in the kinetic interaction procedure, as well as the apparent equilibrium constants, were obtained.
Keywords: Ractopamine; Bovine serum albumin; Molecular spectroscopy; Interaction; Multivariate curve resolution-alternating least squares;

Application of a bridging ELISA for detection of anti-erythropoietin binding antibodies and a cell-based bioassay for neutralizing antibodies in human sera by Sug Kyun Shin; Sung Kyu Ha; Kang Wook Lee; Tae-Hyun Yoo; Sung-Ro Yun; Se-Hee Yoon; Sung-Jo Kim; Soo-Kyeong Lee; Tae-Hwe Heo (289-293).
Although erythropoietin (EPO)-related pure red-cell aplasia (PRCA) is a rare disorder, attention still needs to be paid because underline mechanism of EPO immunogenicity is various and controversial. Among several assay systems for screening of anti-EPO binding antibodies (Abs), we adopted and setup the bridging ELISA using streptavidin-coated plate. To test their neutralizing activities, cell-based neutralizing (NT) bioassay was setup. When we analyzed serum samples by using these two assays, we found two positive results in the two samples. In the sample 1, 411.9 ng/ml of anti-EPO Abs were found and neutralizing activity of 36.2% at 1:5 serum dilution was detected. In the sample 2, 40.5 ng/ml of anti-EPO Abs were found and neutralizing activity of 96.7% was detected. Our results indicate that the higher anti-EPO antibody (Ab) level in a serum does not always lead to the stronger neutralizing activity. This report gives crucial consideration to the needs of establishing clear criteria to link various assay parameters with the onset of PRCA and its progression.
Keywords: Erythropoietin; Pure red-cell aplasia; Bridging ELISA; Neutralizing bioassay; Immunogenicity;

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate by Rebeka Toporišič; Anita Mlakar; Jernej Hvala; Iztok Prislan; Lucija Zupančič-Kralj (294-299).
Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC–MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 μg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 μg/mL and LOQ was 0.5 μg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC–MS/MS n , Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity.
Keywords: Enalapril maleate; Forced degradation; Degradation impurities; HPLC; UPLC–MS; MMPP oxidation;

Interactions of human serum albumin with meloxicam by Lilianna Trynda-Lemiesz; Katarzyna Wiglusz (300-304).
Human serum albumin (HSA) is the most prominent protein in plasma. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme, bilirubin and drugs. Here, we report the effect of new generation, non-steroidal anti-inflammatory drug (NSAID) meloxicam on the albumin conformation and ligand binding. In the present work the interaction of meloxicam with HSA in aqueous solution at physiological pH has been investigated through circular dichroism and fluorescence spectroscopy. The strong quenching of the fluorescence clearly indicated that the binding of the drug to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. This was confirmed by the destabilization of the warfarin binding site. CD and fluorescence spectroscopic results showed marked reductions (about 40% decrease in the CD Cotton effect intensity, and ∼15% decrease of the fluorescence intensity) in the affinity of albumin for bilirubin upon meloxicam binding. The strong inhibition of warfarin and ANS bound to protein after meloxicam modification compared with aspirin confirms that the binding site of both drugs is not the same.
Keywords: Meloxicam; Aspirin; Human serum albumin; Circular dichroism; Fluorescence;

Clinically used anticoagulant warfarin is usually available as a racemic mixture of S- and R-warfarin that are both metabolized mainly by cytochrome P450 isoenzymes. Determination of warfarin enantiomers and their enantiomeric 7-hydroxywarfarin (7-OH-warfarin) metabolites in the human plasma sample allows probing of cytochrome P450 isoenzyme activities and detection of ingestion of warfarin-containing products for the investigation of unexplained bleeding. The present study aims to develop a sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous detection of S-warfarin, R-warfarin, S-7-OH-warfarin and R-7-OH-warfarin in human plasma. Plasma samples were extracted with mixed-mode cation-exchange (MCX) cartridge with recoveries of greater than 87.0% for all four analytes. The selectivity of 7-OH-warfarin from other monohydroxylated warfarin metabolites such as 4′-, 6-, 8- and 10-hydroxywarfarins using a Chirobiotic V chiral column and multiple reaction monitoring (MRM) was addressed. The developed LC–MS/MS method is simple, specific and has been fully validated with satisfactory accuracy and adequate reproducibility with limit of quantification (LOQ) of 5 ng/ml for all four analytes. The method was successfully applied to analyze the steady state plasma concentrations of the four analytes in 30 patients.
Keywords: S-Warfarin; R-Warfarin; S-7-Hydroxywarfarin; R-7-Hydroxywarfarin; LC–MS/MS;

The pressure to reduce cycle times of sample analysis has made it increasingly important to improve sample throughput during pharmaceutical process development. For ICP-based analyses, sample preparation is often the bottleneck of the entire analytical scheme due to the tedious digestion procedure and lacking a universal diluent for organic compounds. In this work, N,N-dimethylformamide (DMF) was used as a “universal” organic diluent so that the sample preparation can be simplified as a “dilute-and-shoot” procedure. An optimized interface with a commercial membrane desolvation unit was implemented, which enabled the introduction of organic solvents into an ICP-AES without organic loading. Mixed standard solutions of 15 elements (Al, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pd, Pt, Rh, Ru, W, Zn, and Zr), which covered the majority of processing metals routinely monitored in pharmaceutical development, were prepared for the study and stability of each element in a multi-element DMF solution was investigated. It was found that the addition of a stabilizing agent (EDTA) was necessary to ensure that all the elements at concentrations of 0.10–0.50 μg/mL remained physically stable in solution (recovery better than 95%) for 2 weeks. It was also important to use an internal standard (yttrium) in order to compensate for signal drift and matrix effects from different sample matrices. A 2–10-fold increase of sensitivity (due to enhanced analyte transport efficiency) and acceptable levels of precision (RSD < 3%) and recoveries (91–111%) were achieved. The LOQs of all 15 elements were less than 10 μg/L in the solution, which translates to less than 5 μg/g or μg/mL in pharmaceutical samples tested. This technique would minimize the effort required for sample preparation, thus reducing the cycle time by approximately 60–90% in the entire analytical scheme for samples that are difficult to be dissolved in nitric acid. This will provide opportunities for a new level of sample handling and automation for metal analysis in pharmaceutical process development.
Keywords: Inductively coupled plasma-atomic emission spectrometry (ICP-AES); Metal analysis; Pharmaceutical; Sample preparation; Organic diluent;

During development of the residual solvent method using headspace-GC for a drug substance, an unexpected peak was observed in the chromatography. GC–MS analysis confirmed the unknown peak identity as isobutylene. An understanding of the source of the isobutylene was required in order to develop appropriate impurity and residual solvent control strategies for the drug substance. The experiments performed to determine the source of the isobutylene peak observed in the headspace-GC chromatography and how the tert-butoxycarbonyl (BOC) de-protection step used in the drug substance synthesis contributes to its observation are discussed.
Keywords: Headspace-GC; Residual solvent analysis; Isobutylene; tert-Butoxycarbonyl (BOC) protection; Drug substance;

Acid and base degraded products of ketorolac by Margherita Salaris; Maria Nieddu; Nicola Rubattu; Cecilia Testa; Elvira Luongo; Maria Grazia Rimoli; Gianpiero Boatto (320-322).
The stability of ketorolac tromethamine was investigated in acid (0.5 M HCl) and alkaline conditions (0.5 M NaOH), using the same procedure reported by Devarajan et al. . The acid and base degradation products were identified by liquid chromatography–mass spectrometry (LC–MS).
Keywords: Ketorolac; Acid and base stability; Degradation products; LC–MS;