Orthogonal extraction/chromatography and UPLC, two powerful new techniques for bioanalytical quantitation of desloratadine and 3-hydroxydesloratadine at 25 pg/mL

Validation of the bioanalytical method for determination of desloratadine and 3-hydroxydesloratadine was conducted using ultra high pressure liquid chromatography (UPLC) in conjunction with mix mode solid phase extraction. The dynamic range of the assay was from 0.025 ng/mL to 10 ng/mL using 96-well solid phase extraction. On an UPLC system, the inter-run accuracy was better than 94.7% for desloratadine (n = 18) and 94.0% for 3-hydroxydesloratadine (n = 18). The between-run precision (%CV) ranged from 2.6% to 9.8% for desloratadine (n = 18) and 3.1% to 11.1% for 3-hydroxydesloratadine (n = 18). The limit of quantitation represented 0.478 pg and 0.525 pg of extracted material injected on-column for desloratadine and 3-hydroxydesloratadine, respectively. The total run time was slightly over 2 min per sample. The approach of orthogonal extraction/chromatography and UPLC significantly improves assay performance while also increasing sample throughput for drug development studies.     

A Chromatographic Determination of Aripiprazole using HPLC and UPLC: A Comparative Validation Study

A simple, precise, and accurate isocratic reversed-phase (RP) stability-indicating HPLC assay method was developed and validated for determination of Aripiprazole in bulk and solid pharmaceutical dosage form. A reversed-phase C8 (250×4.0 mm, 5 μm particle size) column for HPLC and C8 (50×2.1mm, 1.7 μm particle size) for UPLC method in isocratic mode was used. The mobile phase consists of acetonitrile: 20 mM ammonium acetate (90:10, v/v), flow rate was set at 1.0 ml/min and 0.250 ml/min for HPLC and UPLC, respectively and the detection was performed for both methods were at 240 nm. Further the validation of both developed method was performed and subsequently compared to prove its better applicability.     

Determination of bergamottin in human plasma after grapefruit juice ingestion by an UPLC/MS/MS method

Bergamottin was identified as a cause of pharmacokinetic interaction with grapefruit juice intake and as a physiologically active substance involved in lipolysis. However, the quantification method on concentrations of bergamottin in systemic circulation has not been well established. The aim of this study was to develop a simple, sensitive and high-throughput determination system for bergamottin in human plasma using an ultra performance liquid chromatography (UPLC)-MS-MS method. The UPLC system equipped with a UPLC BEH C18 column (2.1 x 50 mm, 1.7 microm) and an ESI prove was appropriate for detection of bergamottin. As a result, a primary product ion (m/z = 203) from precursor ion of bergamottin (m/z = 339) was observed. Plasma from a human volunteer who consumed grapefruit juice one hour before the time of blood sampling, was measured with the UPLC/MS/MS system. The determination of plasma-bergamottin was performed with the highest sensitivity presently available. In conclusion, we succeeded high performance bergamottin-determination in human plasma after grapefruit juice ingestion. The procedure can be usefulto clarify pharmacokinetic and pharmacodynamic characteristics of bergamottin.     

Evaluation of a Dual-Wavelength Size Exclusion HPLC Method With Improved Sensitivity to Detect Protein Aggregates and Its Use to Better Characterize Degradation Pathways of an IgG1 Monoclonal Antibody

The evaluation of a dual wavelength size exclusion high performance liquid chromatography (DW-SE-HPLC) method with improved sensitivity to detect aggregates in a high concentration IgG1 monoclonal antibody formulation is presented. This technique utilizes ultraviolet detection at two different wavelengths to monitor the levels of monomer, aggregate, and fragments and was shown to have improved sensitivity for the detection aggregates and fragments compared to light scattering (LS) detection. After assay optimization including the use of column conditioning, the limit of quantitation for aggregates was determined to be 0.04% with essentially complete recovery of aggregates from the column (> 99.5%). The DW-SE-HPLC method was used to evaluate the level of protein aggregates generated by different environmental conditions such as exposure to elevated temperatures/acidic pH or intense light. The detection and characterization of protein aggregates by DW-SE-HPLC was compared with an orthogonal biophysical technique (sedimentation velocity analytical ultracentrifugation, SV-AUC). A good overall correlation was observed for levels of monomer, aggregates (dimer and multimers), and fragments as measured by the two analytical techniques (e.g., 6.0% vs. 5.3% and 14% vs. 11% for dimeric aggregates generated by elevated temperature/acidic pH and light exposure, respectively). The stability profile of a high concentration IgG1 monoclonal antibody formulation was investigated under stressed storage conditions (40 °C over 3 months) using the DW-SE-HPLC method including the loss of monomeric species with the concomitant accumulation of both aggregates and fragments. The nature and composition of the aggregates (primarily noncovalent dimers) and fragments (primarily loss of Fab from an intact IgG1) formed during storage were further characterized by a combination of LS measurements and mass spectroscopy analysis of deglycosylated IgG1 samples isolated by preparative SE-HPLC. The combination of DW-SE-HPLC, SV-AUC, LS, and mass spectroscopy results provided a detailed overall understanding the monomer, aggregate, fragment degradation pathway(s) for a high concentration IgG1 monoclonal antibody formulation during storage.     

High-Performance Liquid Chromatographic (HPLC) Determination of Lobenzarit in Plasma and Its Application to a Bioavailability Study in Beagle Dogs

A method for the quantitative determination of lobenzarit (2-[(2-carboxyphenyl)amino]-4-chlorobenzoic acid) in dog plasma by high-performance liquid chromatography with UV detection (308 nm) is described. Plasma samples (200 µl) were treated with acetonitrile and centrifuged, and the clear supernatant injected onto a reversed-phase phenyl column. The method achieved a limit of quantitation of 0.5 µg/ml in plasma, and the response was linear to 100 µg/ml. Comparing a solution and a tablet formulation given to beagle dogs, the assay demonstrated that the solution formulation was slightly more bioavailable and yielded a more variable absorption rate. The elimination of lobenzarit from plasma followed a biexponential time course, with an apparent terminal disposition half-life of between 5.8 and 10.7 hr.     

Capillary gas chromatographic analysis of trichlorfon in dosed feed formulations

A method was developed for the analysis of trichlorfon as the intact molecule from feed formulations of the chemical. The method involved isolation of trichlorfon by a simple extraction procedure. Subsequent detection and quantitation of the trichlorfon was by gas chromatography with a DB-1 fused-silica wide-bore capillary column and flame photometric detection. Automated on-column injections were performed. The method was linear in the range 5-75 ng injected on-column and had an average recovery of 86%. The limit of detection for trichlorfon in feed was 1 ppm. The applicability of the method was tested by determining the stability of trichlorfon over a 21-day period in a feed blend of trichlorfon at the 50-ppm concentration level. Mass spectrometric analyses were performed to confirm that trichlorfon could be quantitated as the intact molecule.     

Validation and application of a GC-MS method for determining soman concentration in rat plasma following low-level vapor exposure

A method for determining the chemical warfare agent soman (GD) in rat plasma has been validated and applied to low-level inhalation exposure studies currently being conducted. This method utilizes a fluoride ion-based regeneration assay with isotope dilution followed by large volume injection gas chromatography with ammonia chemical ionization mass spectrometric detection. Following sample preparation by solid phase extraction, chromatographic separation was achieved using a 14% cyanopropylphenyl/86% dimethyl polysiloxane capillary column with a total run time of 18.16 min. Soman and the deuterated isotope ((2)H(4)-soman) internal standard were detected using the selected ion monitoring mode and quantitated using the ammonia adduction ratio of m/z ions 200/204. A reproducible linear relationship was obtained for the quantitative concentration range of 10 pg on-column to 1000 pg on-column (r(2) = 0.9995) for standards in ethyl acetate with a detection limit of 5.65 pg on-column, and an average recovery of 93% in plasma. This sensitive method was successfully applied to the analysis of soman in rat plasma immediately post-exposure, resulting in the construction of dose-response plots.     

Development and validation of UPLC method for determination of primaquine phosphate and its impurities

With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations. Recently, commercially available ultra-performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of fast chromatographic separations. In this work, a new isocratic reverse phase chromatographic method was developed using UPLC for primaquine phosphate bulk drug. The newly developed method is applicable for assay and related substance determination of the active pharmaceutical ingredient. The chromatographic separation of primaquine and impurities was achieved on a Waters Acquity BEH C18, 50 x 2.1mm, 1.7 microm column within a short runtime of 5 min. The method was validated according to the regulatory guidelines with respect to specificity, precision, accuracy, linearity and robustness. Forced degradation studies were also performed for primaquine phosphate bulk drug samples to demonstrate the stability indicating power of the UPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity.     

Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part I: isocratic separation.

Liquid chromatography (LC) is considered to be the gold standard in pharmaceutical analysis. Today, there is a need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in few minutes or even seconds. The present work describes a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC. In order to carry out fast separations, short columns (20-50mm) packed with small particles (3.5 and 1.7 microm) were used and their chromatographic performance was compared to that of a conventional column (150 mm, 5 microm). For that purpose, an optimized LC system was employed to limit extra-column volumes which can have a dramatic impact on efficiency and resolution. This paper reports the fundamental equations used for transferring an isocratic chromatographic separation performed with a given column geometry and chemistry to a smaller column packed with similar or identical stationary phase, without influence on chromatographic performance. For this purpose, the flow rate and the injected volume need to be adapted. The effect of column length and particle size reduction on chromatographic resolution and analysis time was described for an isocratic separation. Using the method transfer equations, it is possible to predict the new conditions to be used, for fast and ultra-fast separations. In this work, ultra-fast separations were achieved thanks to a new generation of instrumentation (ultra performance liquid chromatography, UPLC) which uses simultaneously short column packed with sub-2 microm particles and ultra-high pressure (up to 1000 bar). This work demonstrates an analysis time reduction up to a factor 12, compared to a conventional LC separation, without affecting the quality of separation. Therefore, the complete resolution of a pharmaceutical formulation was achieved in only a few seconds.     

Application of a high-throughput screening procedure with PEG-induced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies

Protein solubility is a critical attribute in monoclonal antibody (mAb) formulation development as insolubility issues can negatively impact drug stability, activity, bioavailability, and immunogenicity. A high-throughput adaptation of an experimental method previously established in the literature to determine apparent protein solubility is described, where polyethylene glycol (PEG) is used to reduce protein solubility in a quantitatively definable manner. Utilizing an automated, high-throughput system, an immunoglobulin G (IgG)1 mAb in a variety of buffer conditions was exposed to increasing concentrations of PEG and the amount of protein remaining in solution was determined. Comparisons of PEG(midpt) values (the weight% PEG in solution required to decrease the protein concentration by 50%) to extrapolated values of apparent protein solubility (in the absence of PEG) were performed. The determination of PEG(midpt) by using sigmoidal curve fitting of the entire data set was shown to be the most precise and reproducible approach for use during high-throughput screening experiments. The high-throughput PEG methodology was then applied to the screening of different formulations to optimize relative protein solubility profiles (weight% PEG vs. protein concentration and their corresponding PEG(midpt) values) in terms of solution pH and buffer ions for both human and chimeric IgG1 mAbs. Other comparisons included evaluating relative solubility profiles of an IgG1 mAb produced from different cell lines (Chinese hamster ovary vs. murine) as well as for different IgG1 mAbs (produced from the same cell line) in a series of formulation buffers. Based on these comparisons, it was concluded that rapid, high-throughput determinations of relative protein solubility profiles can be used as a practical, experimental tool to compare mAb preparations and to rank order buffer and pH conditions during formulation development.     

白术的UPLC指纹图谱

目的 建立白术的超高效液相色谱指纹图谱方法。方法 色谱柱为ACQUITY UPLC BEH C18(1.0 mm×50 mm,1.7 μm),乙腈-水二元梯度洗脱模式,流速为0.1 mL·min^-1,紫外检测波长242 nm,建立了32批不同产地白术样品的UPLC指纹图谱。结果 白术药材有32个共有峰,多数峰可达到较好的分离,各批次白术药材间共有峰的相对保留时间RSD均<1.0%,药材间相似度均>90%。结论 本方法简便、可靠,较之HPLC具有更高的分辨率和灵敏度,极大地缩短了分析时间,建立的共有模式较为可靠,可用于白术药材真伪鉴别和质量评价的快速分析。     

建立UPLC法测定复方氨基酸胶囊(9-5)中氨基酸的含量

目的通过优化色谱条件建立UPLC法测定复方氨基酸胶囊(9-5)中氨基酸的含量。方法以9种氨基酸对照为外标物,异硫氰酸苯酯为柱前衍生剂,采用ACQUITY UPLCBEH C_(18)(2.1 mm×100 mm,1.7μm)柱,流动相A为0.1 mol/L的醋酸钠溶液(用冰醋酸调整pH至6.50)-乙腈(93∶7),流动相B为乙腈-水(4∶1),检测波长254 nm,流速为0.45 mL/min,柱温36℃。结果复方氨基酸胶囊(9-5)中9种氨基酸出峰时间与对照品出峰时间一致,其他物质无干扰;仪器精密度RSD为0.5%~1.2%,中间精密度RSD为1.3%~1.9%;各氨基酸的溶液浓度与其峰面积线性关系良好(r≥0.999),溶液稳定性RSD为0.6%~1.8%,各氨基酸的平均回收率在95.0%~105.0%之间。含量测定方法比对实验中UPLC法与HPLC法分析结果无明显差异,分析速度明显提高。结论建立的UPLC法高效,快速,灵敏,准确,可用于测定复方氨基酸胶囊(9-5)中氨基酸的含量。     

Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

Miniaturized Assay for Solubility and Residual Solid Screening (SORESOS) in Early Drug Development

Purpose The aim was to develop a miniaturized method for solubility and residual solid screening of drug compounds in aqueous and non-aqueous vehicles in early drug development. Methods Different crystal modifications of caffeine, carbamazepine, and piroxicam were added into 96-well filter plates and solubility was determined in 100 μl of 17 pharmaceutical vehicles. After filtration, drug concentration was determined by Ultra Performance Liquid Chromatography™ (UPLC). Residual solid drug in the filter plates was analyzed by high-throughput (HT) transmission X-ray Powder Diffraction (XRPD). Results HT XRPD analysis revealed solid form conversions of all compounds during solubility determination, e.g., formation of hydrates in aqueous vehicles (caffeine, carbamazepine, piroxicam) or conversion of a metastable crystal form to the stable form (caffeine). Drug solubility was strongly dependent on the crystal modifications formed during the solubility assay. Conclusions The new assay allows the simultaneous, small scale screening of drug solubility in various pharmaceutical vehicles and identification of changes in solid form. It is useful for the identification of formulations and formulation options in non-clinical and clinical development.     

Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion

An HPLC procedure was validated for determining the purity with respect to the charge variant distribution of the recombinant monoclonal antibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments prior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyroglutamate at the amino-terminus of the light and heavy chains (Fab-pE/Q variants) from the variants resulting from the processing of the carboxy-terminal lysine residues of the heavy chains (Fc-Lys variants). The assay demonstrated good linearity, yielding correlation coefficients of >0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recovery of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the range 2–10% of the total protein. The limit of detection was 0.2 μg and 0.1 μg for Fc and Fab variants, respectively. The repeatability of the assay and intermediate precision had relative standard deviation (RSD) values of <1%. Parameters of the papain digest (time, digest stability, reagent stability, pH and papain vendor) and of the chromatography (mobile phase pH, stability, buffer concentration, and column lot and aging) were evaluated for robustness and determined to be acceptable. Data are presented demonstrating the suitability of the assay for determining the product purity of a recombinant MAb.     

夏枯草中熊果酸含量的UPLC测定

目的：建立夏枯草药材中熊果酸含量的超高效液相色谱法（UPLC）测定方法。方法采用UPLC进行测定,色谱柱为ACQUITY UPLC BEH C18（2.1 mm×100 mm,1.7μm）,以甲醇-0.5%冰乙酸水为流动相进行等度洗脱,流速为0.3 ml/min,柱温30℃,检测波长210 nm。结果熊果酸在0.2461～0.4922 mg范围内呈良好的线性关系（r^2=0.9999）,平均回收率为99.29%,R SD=1.74%,熊果酸在夏枯草中的含量为0.28%～0.53%。结论该方法快速、高效、重现性好,可以为夏枯草药材的质量控制提供更有效的手段。     

UPLC法测定大鼠血浆中Liguzinediol浓度以及动力学研究

目的建立测定大鼠血浆中Liguzinediol浓度的UPLC测定方法,探讨其在大鼠体内的药代动力学。方法血浆样品经甲醇提取后,上清液经N2吹干流动相复溶后注入UPLC分析,色谱柱为UPLC HSS T3柱(2.1 mm×100 mm,1.8μm),流动相为甲醇-水(28∶72,V∶V),流速为0.4 ml.min-1,检测波长278 nm。SD大鼠6只,iv给药10 mg.kg-1后,用UPLC法测定给药后大鼠血浆中Liguzinediol的浓度,利用DAS软件拟合并计算其药代动力学参数。结果 Ligu-zinediol的血药浓度在0.41～52.4 mg.L-1范围内呈线性,提取回收率均>80%,日内、日间精密度<10%,符合生物样品分析要求。大鼠静脉注射10 mg.kg-1的Liguzinediol,其血药浓度(C)-时间(t)曲线呈二室模型,主要药动学参数T12β、AUC(0-∞)、CL分别为1.62 h、18.36 mg.h.L-1、0.71 L.h-1.kg-1。结论该方法操作简便、快速、专属性强,可用于Liguzinediol的药代动力学及成药性研究。     

A retention index library for commonly encountered drugs and metabolites using tri-n-alkylamines as reference compounds, nitrogen-phosphorus detectors, and dual capillary chromatography

The retention index system is a reproducible means for the identification of drugs in toxicology samples analyzed by gas chromatography with flame ionization detectors. Nitrogen-phosphorus (NP) detectors do not respond well to the standard n-alkanes, which lack nitrogen and phosphorus groups and which are used to obtain the Kovats index. A method is presented that creates a retention index library based upon a homologous series of tri-n-alkylamines for a series of 125 drugs and metabolites. Standards were analyzed under temperature programmed conditions by simultaneous dual column chromatography using a 5% phenyl and a 50% phenyl methyl silicone fused-silica capillary column. By increasing the hydrogen and air flows to the NP detectors, the Kovats indices were calculated on the system using the n-alkanes C8 through C32. Tri-n-alkylamine indices calculated under the same conditions were compared to the Kovats indices for 14 drugs on the 5% phenyl column and for 12 drugs on the 50% phenyl column. The correlation was found to be linear. The Kovats indices on the 5% phenyl column were compared to previously reported values and found to be consistent.     

连花清瘟胶囊超声逆流提取液超高效液相色谱指纹图谱研究

目的 采用超高效液相色谱（UPLC）法建立连花清瘟胶囊超声逆流提取液的指纹图谱.方法 采用Acquity UPLC@BEH C18柱（100mm×2.1 mm,1.7 μm）,流动相为乙腈-0.1％磷酸溶液梯度洗脱,流速为0.4 mL/min,柱温为40℃,检测波长为228 nm.结果 建立了连花清瘟胶囊超声逆流提取液的UPLC指纹图谱,共标定了27个共有峰,指认了其中4个共有峰,11批连花清瘟胶囊超声逆流提取液指纹图谱的相似度均在0.991 ～1.000之间.结论 该方法稳定性、重复性好,可为连花清瘟胶囊超声逆流提取液的质量评价提供依据.