<p>Liquid chromatography coupling with mass spectrometry (LC/MS) plays an important role in pharmaceutical characterization because of its ability to separate, identify, and quantify individual compounds from the mixture. Polymer brush layer bonded to the silica surface is designed as a novel stationary phase to improve the LC resolution and MS compatibility. The polymer thickness can be controlled to shield the analyte from interacting with the active silanol on the surface and reduce peak tailing. The functional group of the polymer can be changed to tune the selectivity in different separation modes. </p><p> </p><p>Two projects on LC/MS method development for biomolecule characterization using polymer-shell column are discussed in this work. In the first project, a polymer-shell column is used for disulfide bonds and free thiol subspecies identification, which is a major type of structural heterogeneities in IgG1. Compared with commercial columns, the polymer-shell column is able to resolve the free thiol variants without the presence of trifluoroacetic acid and greatly improve the MS signal. In the second project, a polymer-shell column is used for characterizing the drug-loading profile for antibody-drug-conjugates (ADC) via online LC/MS. The separation employs a mobile phase of 50 mM ammonium acetate to keep the ADC intact, and a gradient of water/isopropanol for ADC elution. MS data show that all ADC species remained intact and native on the column. Positional isomers can be separated and identified with the new method as well. Furthermore, to understand the surface chemistry and protein separation behavior quantitatively, a chromatographic simulation study is performed. The result shows that protein separation in RPLC can be described by a bi-Langmuir adsorption isotherm with mixed-mode retention of strong and weak sites. Smaller fractions and lower equilibrium constant of the strong site, which is the active silanol, give less tailing for protein separation.</p>

<p>Therapeutic proteins are often lyophilized with excipients such as sucrose or trehalose to protect them during manufacturing and achieve a longer shelf-life. Formulation design for therapeutic proteins has been a trial-and-error process, and the mechanisms responsible for the stabilizing effects of excipients are not fully understood. Two proposed theories have been widely accepted: the water replacement theory and the vitrification theory.^1,2The water replacement theory suggests that excipients stabilize protein molecules in the solid state by forming hydrogen bonds that “replace” the hydrogen bonds to water that stabilize the protein in solution, while the vitrification theory asserts that proteins are stabilized by a glassy solid matrix of low mobility and does not require direct interactions between excipient and protein. A better understanding of the interactions between proteins and other components of the lyophilized matrix can facilitate rational formulation design and shorten the time in development. However, most of the analytical methods available can only provide information on the bulk properties of the lyophilized matrix such as moisture content and glass transition temperature (<i>T</i>_g); it has been difficult to measure the interactions between protein and excipient directly, if they exist. In order to characterize the interactions between protein and excipients in a lyophilized matrix with high resolution, a photolytic labeling method was developed in this dissertation, building on previous work in our research group. Photolytic labeling has long been used to identify protein-protein interactions <i>in vivo</i>.^3,4Common types of photo-reaction reagents and their applications are summarized in Chapter 1. The research described in this dissertation utilizes the diazirine functional group, which is activated after UV exposure and undergoes a free radical reaction to form covalent bonds with nearby molecules. The reaction can be used to identify the interactions between excipients and protein or peptide in a solid formulation. Previous studies in our lab have shown that photo-reaction can be applied to lyophilized solids to study protein-matrix properties and interactions in the solid.^5,6This dissertation seeks to further identify photo-reaction products and analyze them in a more quantitative way. </p><p> </p><p>Chapter 2 describes a quantitative analysis of photo-reaction products in solution and lyophilized solids using a model peptide, KLQ (Ac-QELHKLQ-NHCH₃). The purpose of the work in this chapter is to establish a quantitative analytical method for photo-reaction products, enabling studies of peptide-excipient interactions in lyophilized solids. KLQ was derivatized with a bifunctional probe NHS-diazirine (succinimidyl 4,4’-azipentanoate; SDA) at Lys5 to be photo-reactive. The SDA derivatized KLQ (KLQ-SDA) was used to study the photo-reaction products and examine excipient interactions. Identification and quantitation of photo-reaction products of KLQ-SDA was achieved with liquid chromatography mass spectrometry (LC-MS) and reversed phase HPLC (rp-HPLC). Important reaction products such as peptide-excipient adducts and peptide water adducts varied in different formulations. Unexpected reaction products such as unproductive “dead-end” products and peptide-phosphate adducts from buffer salt were also detected and quantified. Together, the photo-reaction products reflected the local environment near Lys5 of the peptide in the solid state. This study has provided a better understanding of photo-reaction with diazirine in the lyophilized solids together with a quantitative description of the local environment near Lys5. </p><p> </p><p>In Chapter 3, the photo-reaction products in lyophilized solids exposed to increasing moisture were analyzed, and the effect of increasing moisture on the local environment near the peptide was examined. Using the analytical method developed in Chapter 2, these studies explored whether peptide-water interactions, as measured by the formation of water adducts formed by photolytic labeling, are linearly correlated with an increase in solid bulk moisture content. Formulations containing the KLQ-SDA peptide were exposed to various relative humidity conditions and photolytic labeling was induced. Solids containing disaccharide excipients behaved differently from those containing amino acids when exposed to the same relative humidity condition, showing different levels of peptide-excipient and peptide-water adducts. With increasing moisture content in the solids, the formation of photo-reaction products did not mimic the pattern of solutions with same composition, indicating differences in the local environment. </p><p> </p><p>An alternative approach to studying lyophilized formulations using photolytic labeling is to incorporate photo-reactive excipients into the solid matrix. In Chapter 4, a new diazirine-labeled photo-excipient, photo-glucosamine (pGlcN), was chemically synthesized and incorporated into formulations of the therapeutic peptide salmon calcitonin (sCT) and compared with the commercially available diazirine-labeled amino acid, photo-leucine (pLeu). The studies in Chapter 4 further compared peptide-excipient interactions at the molecular level with two different photo-excipients, ionizable pLeu and unionizable pGlcN. Changing solution pH prior to lyophilization was expected to change ionic interactions between sCT and pLeu in the solid samples, resulting in different distributions of photo-reactions products; pH-dependent differences were not expected for pGlcN. The results demonstrated that the distribution of photo-reaction products varied with the composition of the formulation and the pH of the solution prior to lyophilization. The photo-reaction products in the pGlcN-containing formulation differed from those pLeu, showing a difference in the interactions of unionizable (pGlcN) and ionizable (pLeu) excipients with sCT in solid samples. </p><p> </p><p>The work in this dissertation has developed photolytic labeling as a tool to study lyophilized peptide formulations, and has provided a more quantitative understanding of the photo-reaction products that are produced from diazirine-labeled peptides or excipients in the solid state. A new photo-reactive excipient has also been presented (pGlcN), which showed different photo-reaction products than a commercially available photo-excipient (pLeu) and is promising for future study. Photolytic labeling for formulation development is still in its early stages, and additional research regarding reaction mechanism and complementary stability studies is needed. Nevertheless, the results presented in this dissertation support continued development of photolytic labeling as a practical method for formulation design and development. </p><p> </p>

Reversed-phase liquid chromatography (RPLC) is a widely used technique for analytical separations but routinely requires empirical optimization. Gaining a better understanding of the molecular reasons for retention may mean more efficient separations with fewer trial and error runs to obtain optimized separations. Vibrationally resonant sum frequency generation (VR-SFG) is a surface specific technique that has allowed for in situ examination of model RPLC stationary phases under various solvent and pressure conditions. In order to improve on past work with model RPLC stationary phases two challenges had to be overcome. First, improved vibrational mode assignments of the C18 stationary phase were needed for proper understanding of this model system. Second, the synthesis of back-surface reference mirrors used in these VR-SFG experiments allowed us to better correct the relative intensities of the various spectral peaks present in typical spectra. After examination of model RPLC systems under various conditions, we have found that these model substrates have a significant amount of interference from nonresonant signal. This interference of resonant and nonresonant signals on fused silica surfaces has not been previously examined and further studies of the model RPLC stationary phase must properly deal with the non-negligible nonresonant interference that is present. We have seen changes in the VR-SFG spectra of these model systems under a variety of conditions including elevated pressure, however the changes are mostly due to nonresonant interference. These spectral changes, although apparently not solely from structural changes, need to be investigated further to better understand the molecular basis of retention in model RPLC systems.

My work has focused on a variety of different types of diamond-based, core-shell particles. These particles are formed with inert cores and poly(allylamine)/nanodiamond shells. Their intended purpose is to form an LC stationary phase that is stable from pH 1 – 14 and at elevated temperatures. At the beginning of my studies, the particles that had been made in the Linford laboratory were pH stable, but irregular and had poor mechanical stability. Since that time, I have worked to improve the particles by using more spherical zirconia and carbon cores, and I have improved their mechanical stability via chemical crosslinking with epoxides. I have performed van Deemter and van’t Hoff analyses to understand the properties of these columns. Efficiencies greater than 100,000 N/m are routinely achieved with these carbon/nanodiamond-based phases. In addition I contributed to two patents that show innovations in diamond functionalization. My contributions involved reduction of an oxidized diamond surface with LiAlH4 prior to functionalization with isocyanates. I also wrote some application notes for the Flare mixed-mode column, which was recently introduced to the market and contains particles comprised of a carbon core and a polymer/nanodiamond shell. These application notes show the gradient separations of four essential oils (lavender, melaleuca, peppermint and eucalyptus), and the isocratic separations of various triazine herbicides and a mixture of β2-agonists and amphetamines.This dissertation contains the following sections. Chapter 1 is a review of liquid chromatographic history and theory. It also includes a history of the use of diamonds in liquid chromatography. Chapter 2 is a study on a glassy carbon core - polymer/nanodiamond shell particle made in our laboratory. Stability studies at pH 11.3 and 13 were performed and different analytes were retained and/or separated on the column. Chapter 3 is a study performed on the Flare mixed-mode column. Separations of tricyclic antidepressants, β2-andrenergic receptor agonists, and linear chain alkylbenzenes were demonstrated with this phase. Van Deemter and van’t Hoff studies were also performed to probe the efficiency and selectivity of this column with different classes of analytes. Chapter 4 chronicles, via SEM and van Deemter analysis, the improvements that have taken place in our column after many iterations of improved synthetic methods and new materials. These include better particle uniformity, particle stability, and column efficiency. Three different carbon cores were analyzed, each better than the previous one. Appendices 1 – 6 are application notes published by Diamond Analytics of β2-andrenergic receptor agonists and amphetamines, triazine herbicides, and lavender, melaleuca, eucalyptus and peppermint essential oils. Appendices 7 and 8 are patents that contain ideas and research contributed by the author.

No description available.

No / Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

No / Surfactants are commonly used as cleansing agents and yet there are concerns that they may also have a role in skin irritation. The lack of suitable methods for the quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reversed-phase liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulfate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. The detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm(2). LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm(2). These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and to the development and optimisation of new consumer wash products. / EPSRC-DTA award / School Life Sciences

Foram desenvolvidas metodologias de cromatografia a líquido de fase reversa baseadas em injeção sequencial (SIC) e em cromatografia a líquido de alta eficiência (HPLC) para determinação de triclosan em amostras de produtos de higiene pessoal e em estudos de adsorção em argilominerais naturais e modificados, visando determinar parâmetros de adsorção de triclosan frente a alguns de seus metabólitos. A determinação de triclosan em enxaguadores bucais foi realizada por SIC com eluição isocrática usando fase móvel constituída por acetonitrila: tampão fosfato de trietilamina 70 mM pH 3,5 na proporção 70:30 (v v-1), obtendo-se limites de detecção e de quantificação de 0,22 e 0,72 mg L-1, respectivamente. Taxas de recuperação entre 96 e 98% foram obtidas da aplicação a amostras reais, sendo que os resultados obtidos pelo método proposto não apresentaram evidências de diferenças estatisticamente significativas em comparação a uma metodologia de referência baseada em HPLC com coluna empacotada. A separação de triclosan (TCS), 2-clorofenol (2-CP), 2,4-diclorofenol (2,4-DCP), 2,4,6-triclorofenol (2,4,6-TCP), 2,3,4-triclorofenol (2,3,4-TCP) e metiltriclosan (MTCS) foi estudada por SIC, obtendo-se a separação de TCS, 2-CP, 2,4-DCP e 2,4,6-TCP com duas etapas de eluição isocrática, a primeira delas com fase móvel 60:40 (v v-1) metanol: tampão acetato de amônio 20 mM (pH 5,5) seguida de eluição com fase móvel 70:30 (v v-1) metanol : tampão acetato de amônio 20 mM (pH 5,5). Nesse caso, os isômeros 2,4,6-TCP e 2,3,4-TCP coeluem. Metiltriclosan, o menos polar desses compostos, pode ser separado de TCS com etapas subseqüentes de eluição. Os métodos foram aplicados para estudar a adsorção de triclosan e seus metabólitos 2,4-DCP, 2,4,6-TCP e metiltriclosan em montmorilonita homoiônica (K+) e modificada com sal de hexadeciltrimetilamônio (HDTMA), observando-se forte adsorção de triclosan e metiltriclosan em comparação a 2-CP, 2,4-DCP e 2,4,6-TCP. A incorporação de HDTMA no argilomineral causou significativo aumento da capacidade de adsorção desses metabólitos, determinada a partir do ajuste dos dados experimentais à equação linearizada de Langmuir, observando-se que a ordem de adsorção é 2,4,6-TCP > 2,4-DCP > 2-CP / Reversed-phase liquid chromatography methodologies based on sequential injection (SIC) and high performance liquid chromatography (HPLC) have been developed for determination of triclosan in samples of personal hygiene products and in studies of adsorption on natural and modified clay minerals aiming to determine kinetic and thermodynamic parameters of adsorption of triclosan in comparison with some of its metabolites. The determination of triclosan in oral rinses with SIC was performed by isocratic elution using a mobile phase of acetonitrile : 70 mM triethylamine phosphate buffer pH 3.5 at the ratio 70:30 (v v-1), obtaining limits of detection and quantification of 0.22 and 0.72 mg L-1, respectively. Recovery rates between 96 and 98 % were obtained from the application to commercial samples, and the results obtained by the proposed method showed no evidence of statistically significant differences compared to the reference methodology based on HPLC with packed column. The separation of triclosan (TCS), 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4 trichlorophenol (2,3,4-TCP) and methyltriclosan (MTCS) was studied by SIC, resulting in the separation of TCS, 2-CP, 2,4-DCP and 2,4,6-TCP with two isocratic elution steps, the first of them with a mobile phase 60:40 (v v-1) methanol: 20 mM ammonium acetate buffer (pH 5.5) followed by elution with 70:30 (v v-1) mobile phase of methanol : 20 mM ammonium acetate buffer (pH 5.5). In this case, the isomers 2,4,6-TCP and 2,3,4-TCP coeluted. Methyltriclosan, the less polar of these compounds, can be separated from TCS with subsequent elution steps. The methods were applied to study the adsorption of triclosan and its metabolites 2-CP, 2,4-DCP, 2,4,6-TCP and methyltriclosan on homoionic montmorillonite (K+) as well as in hexadecyltrimethylammonium salt (HDTMA) modified montmorillonite, noticing a stronger adsorption of triclosan and methyltriclosan compared with 2-CP, 2,4-DCP and 2,4,6-TCP. Incorporation of HDTMA in the clay mineral caused significant increase in adsorption capacity of these metabolites. This capacity was determined by fitting the experimental data to the linearized Langmuir equation. The adsorption order was 2,4,6-TCP > 2,4-DCP > 2-CP.

In this study, a reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed to separate and determine Pt and Pd after formation of their chelates with N,N-diethyl-N&#039 / -benzoylthiourea (DEBT). With the aim of reducing the number of steps in treating the samples, the method developed does not require the elimination of excess chelating reagent before the analysis of metal chelates. The different physical and chemical parameters affecting separation were examined in details. The whole analysis was completed on a C18 column in 16 min at 280 nm, with the mobile phase of acetonitrile-methanol-water (80:10:10, v:v:v) containing 0.20 mol l-1 pH 5.0 acetate buffer at a flow rate of 0.8 ml min-1. Detection limits of the method, based on 3s, were found as 14.2 ug l-1 for Pd and 0.77 mg l-1 for Pt using a 20-ul sample loop. Reproducibility of the method for ten repeated measurements was found as 2.36 % for 0.60 mg l-1 Pd and 2.58 % for 10.0 mg l-1 Pt as % RSD. The proposed method is a rapid, simple and highly selective method for the simultaneous determination of Pt and Pd by HPLC without the need for any interference elimination process.

Botulinum toxin type A (BTTA) is a polypeptide with 1296 amino acids and its used in some pathologies like hyperhidrosis and migraine, but the cosmetic area is the most important application. BTTA is produced from broth-culture synthesized by the Clostridium botulinum as a single peptide with 150 kDa. Reversed-phase liquid chromatography (RP-LC) method was developed and validated for the assessment of botulinum toxin type A in biopharmaceutical formulations. A RP-LC method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.8, and the mobile phase B was acetonitrile, run isocratically at flow rate 0.3 mL/min. Cromatographic separation was obtained with retention time of 11.4 min, and was linear over the concentration range of 0.2-100 IU/mL (r2 = 0.9999) with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.04 and 0.16 IU/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.31% with bias lower than 0.80%. The method was correlated with in vivo and in vitro bioassays. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p <0.05) compared to intact molecule. The validated methods were applied for the determination of BTTA in biopharmaceutical-derived products, giving mean values of the estimated content/potencies 1.16% lower compared to the in vivo bioassay. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure efficacy of the biotechnology-derived product. / A toxina botulínica tipo A (BTTA) é um polipeptídio constituído de 1296 aminoácidos e é recomendada para patologias, como hiperhidrose e enxaqueca, mas é especialmente usada como cosmético. A toxina botulínica é produzida através do processo de fermentação bacteriana e após sucessivas etapas de purificação resulta em uma proteína com 150 kDa responsável pelo seu efeito biológico. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de potência de BTTA em formulações de produtos biofarmacêuticos. No método foi utilizada coluna Zorbax 300 SB C18 (150 mm x 4,6 mm d.i.), mantida a 45 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,8, e a fase móvel B por acetonitrila, eluídas em vazão isocrática de 0,3 mL/min. O método utilizou detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida em 11,4 min, sendo linear na faixa de concentração de 0,2-100 UI/mL (r2 = 0,9999). Os limites de detecção e quantificação foram 0,04 e 0,16 UI/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,31 com bias inferior a 0,80. O método validado foi correlacionado com bioensaios in vivo e in vitro. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais apresentaram diferença significativa em relação a forma intacta (p <0,05). O método proposto foi aplicado para avaliação da potência de BTTA em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio in vivo, observando-se diferença das médias de teor/potência de 1,16% inferior para o método cromatográfico. Contribuíu-se assim para estabelecer procedimentos que aprimoram a caracterização ou o controle da qualidade, garantindo a segurança e eficácia do produto biotecnológico.