A facile fabric phase sorptive extraction method for monitoring chloramphenicol residues in milk samples.

Determination of pharmaceutical active molecules in the biological matrices is crucial in various fields of clinical and pharmaceutical chemistry, e.g., in pharmacokinetic studies, developing new drugs, or therapeutic drug monitoring. Chloramphenicol (CP) is used for treating bacterial infections, and it's one of the first antibiotics synthetically manufactured on a large scale. Fabric phase sorptive extraction (FPSE) was used to determine Chloramphenicol antibiotic residues in milk samples by means of validated HPLC-DAD instrumentation. Cellulose fabric phases modified with polyethylene glycol-block-polypropylene glycol-block-polyethylene glycol triblock copolymer was synthesized using sol-gel synthesis approach (Sol-gel PEG-PPG-PEG) and used for batch-type fabric phase extractions. Experimental variables of the FPSE method for antibiotic molecules were investigated and optimized systematically. The HPLC analysis of chloramphenicol was performed using a C18 column, isocratic elution of trifluoroacetic acid (0.1%), methanol, and acetonitrile (17:53:30) with a flow rate of 1.0 mL/min. The linear range for the proposed method for chloramphenicol (r2 > 0.9982) was obtained in the range of 25.0-1000.0 ng/mL. The limit of detections (LOD) is 8.3 ng/mL, while RSDs% are below 4.1%. Finally, the developed method based on FPSE-HPLC-DAD was applied to milk samples to quantitatively determine antibiotic residues.

Hydrogen Peroxide Treatment of Softwood-Derived Poly(Ethylene Glycol)-Modified Glycol Lignin at Room Temperature.

Recently, a large-scale production system of softwood-derived poly(ethylene glycol) (PEG)-modified glycol lignin (GL) was developed to produce high-quality lignin derivatives with substantially controlled chemical structures and attractive thermal properties. In this study, the further upgrading of GL properties with carboxy functionalization was demonstrated through the room-temperature hydrogen peroxide (H2O2) treatment with the mass ratio of H2O2 to GL, 1:1 and 1:3, for 7 d. The changes in the chemical structure, carboxy group content, molecular weight, and thermal properties of the insoluble portions of partially oxidized glycol lignins (OGLs) were then investigated. Nuclear magnetic resonance and thioacidolysis data revealed that the oxidative functionalization involved the cleavage of ß-O-4 linkages and the oxidative cleavage of guaiacyl aromatic rings into muconic acid-type structures. This was validated by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and potentiometric titration. Overall, the results suggested that the varying outcomes of carboxy group content (0.81-2.04 mmol/g OGL) after 7-d treatment depended on the type of the GL origin having varying amounts of the retained native lignin structure (e.g., ß-O-4 linkages), which were prepared from different source-wood-meal sizes and PEG molecular masses.

Photoresponsive Hydrogels for Studying Mechanotransduction of Cells.

Hydrogels are important platform materials for in vitro cellular studies. Mechanistic studies on durotaxis, the directional movement of a cell affected by a spatial gradient of stiffness of the underlying substrate, requires materials such as polyacrylamide, polyethylene glycol, or PDMS, in which the stiffness can be controlled in a spatiotemporal manner. Here, we describe the synthesis of an o-nitrobenzyl-based photocleavable cross-linker and its incorporation into a polyacrylamide hydrogel to render it photoresponsive. Precise control over the physical properties of the gel allows observation of glioblastoma durotaxis under surface stiffness conditions relevant to the actual brain environment.

PEGylated near-infrared fluorescence probe for mitochondria-targetable hydrogen peroxide detection.

Oxidative stress plays significant roles in the development of various diseases. H2O2 acts as a signaling molecule physiologically or harmful substance pathologically and the mitochondria are one of the most active places for the generation of H2O2. Thus, a new mitochondria-targeted probe 1 for H2O2 detection was synthesized herein, based on D-π-A structure with a large Stokes shift (150 nm) due to its ICT process. To improve its water solubility and sensitivity, probe 2 with PEG chain and probe 3 with two responsive boronated groups were then designed based on the structure of probe 1. As a result, the fluorescence intensity of probe 2 was far higher than that of probe 1 and probe 3 not only in vitro experiment but in cell imaging study with a larger linear range and signal-to-noise ratio, rendering it the best probe for further exogenous and endogenous H2O2 detection in Hela cells.

Confocal Raman spectroscopy at different laser wavelengths in analyzing stratum corneum and skin penetration properties of mixed PEGylated emulsifier systems.

Emulsifier mixtures are widely used in cosmetics and pharmaceutics and thus, brought extensive studies for their performances on skin applications. PEG-20cetyl ether (C20) is recently proposed to induce skin irritation and is of interest to study its skin interactions when mixed with other emulsifiers. PEG-2oleyl ether (O2) and PEG-20stearyl ether (S20) are selected and in specific, 50 mM of C20, O2, S20 as well as Mix1 (50 mM C20 mixed with 50 mM O2) and Mix2 (50 mM C20 mixed with 50 mM S20) solutions were applied on skin samples. Confocal Raman spectroscopy (CRS) analyses of stratum corneum (SC) thickness and SC lipid content were performed after 4 h skin treatments. In parallel, skin penetration properties were also evaluated via CRS by applying procaine solutions with/without emulsifiers on skin samples for 24 h. In terms of the CRS measurements, two excitation wavelengths of 532 nm and 785 nm are both utilized in this study and we secondly aimed to compare their results and suitability in SC and skin analyses. Based on the experimental observations, comparable results are obtained by using both excitation wavelengths of 532 nm and 785 nm demonstrating their suitability in analyzing SC and skin samples. Thereinto, 785 nm laser wavelength shows the advantage of deeper skin penetration and allows the measurements of fluorescent skin samples; 532 nm laser wavelength enables simple measurement performance without substrate and coverslip interference. With regards to the results of emulsifier mixtures, the addition of S20 and O2 reduced the skin interactions and penetration enhancing ability of C20, giving us the hint to build milder systems with emulsifier mixtures. Besides, the CRS results of stronger skin interruption were also correlated with the higher critical micelle concentration (CMC) values of emulsifiers and their mixtures, which may provide evidence in explaining the interactions between emulsifiers and skin.

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Advancing Structure Characterization of PS-80 by Charge-Reduced Mass Spectrometry and Software-Assisted Composition Analysis.

The commercially available Polysorbate 80 (PS-80) is a highly heterogeneous product. It is a complex and structurally diverse mixture consisting of polymeric species containing polyoxyethylenes (POEs), fatty acid esters, with/or without a carbohydrate core. The core is primarily sorbitan, with some isosorbide and sorbitol. Depending on the sources of fatty acids and the degrees of esterification, multiple combinations of fatty acid esters are commonly observed. A number of POE intermediates, such as polyoxyethylene glycols, POE-sorbitans, POE-isosorbides, and an array of fatty acid esters from these intermediates remain in the raw material as well. The complex composition of PS-80 is difficult to control and poses a significant characterization challenge for its use in the pharmaceutical industry. Here, we present a novel solution for PS-80 characterization using ultra high-performance liquid chromatography coupled with charge-reduction high resolution mass spectrometry. Post column co-infusion of triethylamine focused the signal into mainly singly charged molecular ions and reduced the extent of in-source fragmentation, resulting in a simpler ion map and enhanced measurement of PS-80 species. The data processing workflow is designed to programmatically identify PS-80 component classes and reduce the burden of manually analyzing complex MS data. The 2-dimensional graphical representation of the data helps visualize these features. Together, these innovative methodologies enabled us to analyze components in PS-80 with unprecedented detail and shall be a useful tool to study formulation and stability of pharmaceutical preparations. The power of this approach was demonstrated by comparing the composition of PS-80 obtained from different vendors.

A fast screening method for the detection of CERA in dried blood spots.

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 µg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.

In vitro response of vanilla (Vanilla planifolia Jacks. ex Andrews) to PEG-induced osmotic stress.

Drought-induced water stress affects the productivity of the Vanilla planifolia Jacks. ex Andrews crop. In vitro culture technique is an effective tool for the study of water stress tolerance mechanisms. This study aimed to evaluate the morphological, physiological and biochemical response of V. planifolia under in vitro water stress conditions induced with polyethylene glycol (PEG). In vitro regenerated shoots of 2 cm in length were subjected to different concentrations of PEG 6000 (0, 1, 2 and 3% w/v) using Murashige and Skoog semi-solid culture medium. At 60 days of culture, different growth variables, dry matter (DM) content, chlorophyll (Chl), soluble proteins (SP), proline (Pro), glycine betaine (GB), stomatal index (SI) and open stomata (%) were evaluated. Results showed a reduction in growth, Chl content, SP, SI and open stomata (%) with increasing PEG concentration, whereas DM, Pro and GB contents rose with increasing PEG concentration. In conclusion, PEG-induced osmotic stress allowed describing physiological and biochemical mechanisms of response to water stress. Furthermore, the determination of compatible Pro and GB osmolytes can be used as biochemical markers in future breeding programs for the early selection of water stress tolerant genotypes.

Evaluation of Residual Monomers Eluted from Pediatric Dental Restorative Materials.

Unreacted monomers eluted from resin-based restorative materials have been considered a reason of local and systemic adverse reactions. This study was designed to determine the effect of finishing and polishing procedures on the elution of Bis-GMA, TEGDMA, UDMA, and HEMA monomers from compomer and bulk-fill composite resins. Bulk-fill composite (3M ESPE GmbH, Seefeld, Germany) and compomer (Dentsply DeTrey GmbH, Konstanz, Germany) specimens with 3 × 4 mm diameters were prepared. The specimens were randomly divided into two groups, and finishing-polishing procedures were applied only to the experimental groups. Release of residual monomers was analyzed by using High-Performance Liquid Chromatography (HPLC) after 24, 48, and 72 hours. Repeated measures ANOVA and Tukey post hoc tests were used for comparisons. Finishing and polishing procedures had a significant effect on reducing the quantity of UDMA release in the Filtek™ Bulk Fill composite and Bis-GMA, HEMA, and TEGDMA in the Dyract XP compomer (p < 0.05). The restorative materials investigated here are not chemically stable after polymerization, and concentrations of eluted monomers may reach critical toxicity levels even after one restoration placement. Finishing and polishing procedures are mandatory to reduce residual monomers.

Effect of pegylated interferon alfa-2a in HBeAg-negative chronic hepatitis B during and 48 weeks after off-treatment follow-up: the limitation of pre-treatment HBsAg load for the seroclearance of HBsAg.

Hepatitis B virus (HBV) infection is a major public health problem worldwide. The study aimed to evaluate the efficacy of pegylated interferon (Peg-IFN) alfa-2a treatment for seroclearance of HBs antigen (HBsAg) in HBe antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. This retrospective study investigated 16 HBeAg-negative CHB patients who received Peg-IFN alfa-2a weekly for 48 weeks. Thereafter, the patients were followed-up for 48 weeks after the end of therapy. The following criteria were also used for inclusion: HBV-DNA < 5.0 log copies/mL and without nucleot(s)ide analogs. Four HBsAg-positive cases became HBsAg negative. The HBsAg levels of the 4 patients who achieved HBsAg seroclearance were lower significantly than that of the non-seroclearance group (p = 0.007). The mean HBsAg levels in these 4 cases were 68 IU/mL, while the mean HBsAg levels in the non-seroclearance group were 2,114 IU/mL. The mean HBV-DNA levels in the 4 HBsAg seroclearance cases were 2.8 log copies/mL as compared to 3.6 log copies/mL in HBsAg-non-seroclearance cases (p = 0.01). Cases that are HBeAg negative, with HBV-DNA levels < 5 log copies/mL, and HBsAg titers < 120 IU/mL cases may achieve HBsAg clearance with Peg-IFN therapy.

Hybrid MoS2/g-C3N4-assisted LDI mass spectrometry for rapid detection of small molecules and polyethylene glycols and direct determination of uric acid in complicated biological samples.

A novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometric method (MALDI-TOF MS) for determination of highly sensitive small molecular compounds was developed based on molybdenum disulfide nanosheets hybridized with ultrathin graphitic carbon nitride (MoS2/g-C3N4) as the matrix. With this approach, the synergistic effects of MoS2 and g-C3N4 enhance the UV absorption of MoS2/g-C3N4, increase both desorption and ionization efficiency in LDI MS, and induce higher signal-to-noise ratio of analytes when compared with the bare MoS2 and g-C3N4 matrix in the determination of amino acids, antibiotics, neutral oligosaccharides, uric acid, and polyethylene glycols (PEGs). The detection limits of these small molecular compounds are in the ranges 0.1 to 10 µg mL-1, 1*10-3 to 1.0 µg mL-1, 1.0 to 10 µg mL-1, and 2*10-4 µg mL-1, respectively, and the polydispersity index of these PEGs is less than 1.02. Moreover, high salt tolera`nce and homogeneous deposition on the spot results in good reproducibility. The relative standard deviations (RSDs) of shot-to-shot and spot-to-spot (n = 15) of these compounds are less than 10.1% and 12.5%, respectively. With MoS2/g-C3N4, the uric acid in complicated biological samples can be directly determined in combination with LDI-TOF MS. We synthesized MoS2/g-C3N4 nanohybrid as an efficient matrix for MALDI-TOF MS analysis of small molecules as well as quantitative detection of uric acid in human urine.

Highly selective and antifouling electrochemical biosensors for sensitive MicroRNA assaying based on conducting polymer polyaniline functionalized with zwitterionic peptide.

Ultrasensitive and low-fouling microRNA electrochemical biosensors were successfully constructed by introducing thiol-terminated antifouling molecules (peptide sequence, polyethylene glycol, or mercapto alcohol) onto the surface of polyaniline-modified electrodes. For the three kinds of antifouling materials investigated, the newly designed and synthesized peptide exhibited superior antifouling ability to others, and it could effectively reduce the nonspecific adsorption of proteins and even prevent the fouling effect of serum. Compared with microRNA biosensors without antifouling capability, or those modified with polyethylene glycol or mercapto alcohol, the biosensor modified with the designed zwitterionic peptide showed the highest specificity for single-base mismatch, three-base mismatch, and completely complementary microRNAs. Most interestingly, the experimental results indicated that the introduction of antifouling molecules to the sensing interfaces did not significantly change the sensitivity of the biosensor. The strategy of constructing antifouling biosensors based on newly synthesized zwitterionic peptides and conducting polymers can be promisingly extended to the development of other electrochemical sensors and biosensors without encountering biofouling. Graphical abstract Ultrasensitive and low-fouling microRNA electrochemical biosensors were constructed by introducing thiol-terminated antifouling molecules (peptide sequence, polyethylene glycol, or mercapto alcohol) onto the surface of polyaniline-modified electrodes. The biosensor modified with the designed zwitterionic peptide showed the highest specificity amongst four kinds of biosensors.

Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation.

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

Transcriptional response of Bacillus megaterium FDU301 to PEG200-mediated arid stress.

BACKGROUND: For microorganisms on a paper surface, the lack of water is one of the most important stress factors. A strain of Bacillus megaterium FDU301 was isolated from plaques on a paper surface using culture medium with polyethylene glycol 200 (PEG200) to simulate an arid condition. Global transcriptomic analysis of B. megaterium FDU301 grown under normal and simulated arid conditions was performed via RNA-seq technology to identify genes involved in arid stress adaptation. RESULTS: The transcriptome of B. megaterium FDU301 grown in LB medium under arid (15% PEG200 (w/w)) and normal conditions were compared. A total of 2941 genes were differentially expressed, including 1422 genes upregulated and 1519 genes downregulated under arid conditions. Oxidative stress-responsive regulatory genes perR, fur, and tipA were significantly upregulated, along with DNA protecting protein (dps), and catalase (katE). Genes related to Fe2+ uptake (feoB), sporulation stage II (spoIIB, spoIIE, spoIIGA), small acid-soluble spore protein (sspD), and biosynthesis of compatible solute ectoine (ectB, ectA) were also highly expressed to various degrees. Oxidative phosphorylation-related genes (atpB, atpE, atpF, atpH, atpA, atpG, atpD, atpC) and glycolysis-related genes (pgk, tpiA, frmA) were significantly downregulated. CONCLUSION: This is the first report about transcriptomic analysis of a B. megaterium to explore the mechanism of arid resistance. Major changes in transcription were seen in the arid condition simulated by PEG200 (15%), with the most important one being genes related to oxidative stress. The results showed a complex mechanism for the bacteria to adapt to arid stress.

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection.

Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1-1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

Determination of extent of PEGylation using denaturing capillary isoelectric focussing.

Conjugated proteins and enzymes are often formed using N-hydroxysuccinimide (NHS) chemistry, which reacts with free primary amines resulting in a loss of charge and a reduction in isoelectric point (pI). Measurement of the extent of reaction of these conjugates is critical for biopharmaceutical developers. Due to this change in protein charge state, denaturing capillary isoelectric focussing (cIEF) offers a potentially straightforward and convenient approach for extent-of-reaction quantification. Here, we demonstrate the potential of this technique with poly(ethylene glycol) (PEG) conjugates of Erwinia chrysanthemil-asparaginase (ErA). Development of an appropriate sample preparation technique is critical to achieving reproducible cIEF electropherograms, particularly for denaturation-resistant proteins such as ErA, and an emphasis was placed on this during development of the PEG-ErA cIEF method. cIEF electropherograms demonstrating a distribution of PEGylation states in a bell-shaped curve were obtained, and assignment of PEGylation states to these peaks was critical to routine use of the method. The method is sensitive enough to resolve non-lysine adducts of PEG (such as those conjugated to histidine residues) and was shown to give reproducible results over a 2 year period. Biopharmaceutical developers should consider cIEF for extent of reaction monitoring and measurement for conjugates of free amine groups.

Assessment of the Potential and Limitations of Elemental Mass Spectrometry in Life Sciences for Absolute Quantification of Biomolecules Using Generic Standards.

Inductively coupled plasma-mass spectrometry (ICP-MS) has been widely used in Life Sciences for the absolute quantification of biomolecules without specific standards, assuming the same response for generic compounds including complex biomolecules. However, contradictory results have been published on this regard. We present the first critical statistical comparison of the ICP-MS response factors obtained for 14 different relevant S-containing biomolecules (three peptides, four proteins, one amino acid, two cofactors, three polyethylene glycol (PEG) derivatives, and sulfate standard), covering a wide range of hydrophobicities and molecular sizes. Two regular flow nebulizers and a total consumption nebulizer (TCN) were tested. ICP-MS response factors were determined though calibration curves, and isotope dilution analysis was used to normalize the results. No statistical differences have been found for low-molecular-weight biocompounds, PEGs, and nonhydrophobic peptides using any of the nebulizers tested. Interestingly, while statistical differences were still found negligible (96-104%) for the proteins and hydrophobic peptide using the TCN, significantly lower response factors (87-40%) were obtained using regular flow nebulizers. Such differential behavior seems to be related mostly to hydrophobicity and partially to the molecular weight. Findings were validated using IDA in intact and digested bovine serum albumin solutions using the TCN (98 and 100%, respectively) and the concentric nebulizer (73 and 97%, respectively). Additionally, in the case of a phosphoprotein, results were corroborated using the P trace in parallel to the S trace used along the manuscript. This work seems to suggest that ICP-MS operated with regular nebulizers can offer absolute quantification using generic standards for most biomolecules except proteins and hydrophobic peptides.

Simultaneous analysis of filgrastim and pegfilgrastim aggregates by size-exclusion chromatography.

A new size-exclusion high-performance liquid chromatographic (SE-HPLC) method for the simultaneous analysis of filgrastim and pegfilgrastim aggregates was developed and validated. A cross-linked agarose and dextran column was used at ambient temperature and an alkaline sodium phosphate buffer as mobile phase eliminated non-ideal interactions with the stationary phase. The robustness of the method was assessed by varying injection volumes, flow rates and sample vehicles. Other reliability assessments include calibration curve, intra and inter-day precision and accuracy, repeatability of retention times, application to real in-process production samples and column lifetime. The method exhibited linearity over the concentration of 0.02-4 mg/ml range for filgrastim and pegfilgrastim monomer with a correlation coefficient of greater than 0.999. The lower limit of quantification was 0.02 mg/ml and the limit of detection was 0.005 mg/ml. This SE-HPLC technique has been successfully used for several years and more than 10,000 samples.