Use of isopycnic plots to understand the role of density in SFC - I. Effect of pressure variation on retention factors.

This paper aims to demonstrate the effect of pressure variations in modifying analyte retention behavior in SFC. There is a general understanding that in SFC increasing pressure decreases the retention factor (k'), and vice versa. What is not clearly discussed or explained in any recent literature is that these variations can be very different at different operating pressures, temperatures and modifier concentrations. It is important to have a clearer understanding on these variabilities during method development and results analysis. In this paper the nature of k' variation with pressure, at different temperatures and modifier concentrations, will be explained with the help of isopycnic plots of CO2 and CO2+methanol mixtures.

Practical applications of integrated microfluidics for peptide quantification.

BACKGROUND: Increased pressure to obtain more, higher sensitivity data from less sample is especially critical for large peptides, whose already optimized LC-MS methods are heavily challenged by traditional ligand-binding assays. RESULTS: Critical bioanalytical assays were adapted to integrated microscale LC to reduce sample volumes while increasing sensitivity. Assays for teriparatide, glucagon and human insulin and five analogs were transferred from 2.1 mm analytical scale LC to a 150 µm scale system. This resulted in a 15-30 fold overall improvement in sensitivity derived from increased signal to noise, three to six fold reduction in injection volumes, and a two to five fold reduction in sample consumption. CONCLUSION: Integrated microscale LC reduces sample consumption while enabling single picomolar quantification for therapeutic and endogenous peptides.

Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection.

N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

A scaling rule in supercritical fluid chromatography. I. Theory for isocratic systems.

Scaling is regularly done in chromatography either to transfer a successfully designed method of analysis developed in one system to another system, or to scale-up a separation method developed in analytical scale to preparative scale. For liquid chromatography there are well-tested guidelines for scaling, which makes it a routine job. For supercritical fluid chromatography (SFC), on the other hand, neither do we have any well-understood principles behind scaling nor do we know how far the strategies applied in LC could be applicable to SFC. In this article, we have addressed these issues and proposed a rule applicable for scaling isocratic methods between different SFC systems and column dimensions under commonly used operating temperatures and pressures. We have shown that the scale-up and method transfer techniques used in LC can be applied to SFC, provided we ensure that both the original and the target systems in SFC operate at the same average density. The current article will present the theory, discuss the extents of applicability of this rule, and outline its limitations. In an accompanying article implementation of this rule in various practical situations will be presented.

Matrix effects in metabolite quantification for MIST assessment: the impact of phospholipid removal and HPLC column particle size.

BACKGROUND: This Research article investigates the impact of phospholipid removal and high-performance liquid chromatography column particle size on the accuracy of determining the relative abundance of human metabolites using mass spectrometry peak areas in the context of assessing metabolite abundance for Metabolites in Safety Testing assessment. RESULTS/METHODOLOGY: Plasma samples spiked with 20 compounds, representing ten pairs of drugs and metabolites, were prepared using phospholipid removal plates (Ostro™) or standard protein precipitation techniques and analyzed by liquid chromatography-tandem mass spectrometry using high-performance liquid chromatography columns containing either 2.5 or 3.5 µm particles. Removal of phospholipids significantly reduced matrix effects for samples analyzed on the larger particle size columns while preventing phospholipid build up on the analytical columns. In addition, quantitative accuracy and linearity were not affected by phospholipid removal. CONCLUSION: Both sample preparation strategies and column particle sizes should be considered in order to reduce the inaccuracy as a result of matrix effects in assessing metabolite abundance using mass spectrometry peak areas.

Multidimensional LC-MS/MS enables simultaneous quantification of intact human insulin and five recombinant analogs in human plasma.

This work provides a multidimensional method for the simultaneous, direct quantification of intact human insulin and five insulin analogs in human plasma. This investigation solves both the selectivity and sensitivity problems encountered for accurate quantification of insulins in plasma since the former is not possible with conventional assays and the latter with conventional LC-MS/MS. The method uses a mixed-mode SPE and a multidimensional LC method including a solid-core particle column containing an anion exchange stationary phase. Matrix factors for all analogs were calculated in 6 sources of human plasma and CVs of the matrix factors were <15% in all cases supporting the selectivity of the method, while achieving LLOQs of 50-200 pg/mL (1.4-5.6 µIU/mL) for each insulin from 250 µL of human plasma. The average accuracy for the standard curve points in extracted human plasma was 99-100%. Average inter- and intraday accuracies for QC samples were 98% and 94%, respectively. Average inter- and intraday precisions for QC samples were 7.5 and 5.3%, respectively. Patient samples were analyzed in a blind study and results concurred with their diabetes multidosing regimes. The study also demonstrated that the presence of high levels of human insulin and bovine insulin does not interfere with quantification of any of the analyzed analogs. We propose this method for the accurate pharmacokinetic monitoring of diabetic patients, for sport antidoping and forensic toxicology analysis.

High sensitivity LC-MS/MS method for direct quantification of human parathyroid 1-34 (teriparatide) in human plasma.

Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2µm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200µL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide.

High-resolution peptide mapping separations with MS-friendly mobile phases and charge-surface-modified C18.

Ionic analytes, such as peptides, can be challenging to separate by reverse-phase chromatography with optimal efficiency. They tend, for instance, to exhibit poor peak shapes, particularly when eluted with mobile phases preferred for electrospray ionization mass spectrometry. We demonstrate that a novel charged-surface C18 stationary phase alleviates some of the challenges associated with reverse-phase peptide separations. This column chemistry, known as CSH (charged-surface hybrid) C18, improves upon an already robust organosilica hybrid stationary phase, BEH (ethylene-bridged hybrid) C18. Based on separations of a nine-peptide standard, CSH C18 was found to exhibit improved loadability, greater peak capacities, and unique selectivity compared to BEH C18. Its performance was also seen to be significantly less dependent on TFA-ion pairing, making it ideal for MS applications where high sensitivity is desired. These performance advantages were evaluated through application to peptide mapping, wherein CSH C18 was found to aid the development of a high-resolution, high-sensitivity LC-UV-MS peptide mapping method for the therapeutic antibody, trastuzumab. From these results, the use of a C18 stationary phase with a charged surface, such as CSH C18, holds significant promise for facilitating challenging peptide analyses.

Development of a fast method for direct analysis of intact synthetic insulins in human plasma: the large peptide challenge.

BACKGROUND: Intact insulins are difficult to analyze by LC-MS/MS due to nonspecific binding and poor sensitivity, solubility and fragmentation. This work aims to provide a simpler, faster LC-MS method and focuses on solving the above issues. RESULTS: A novel charged-surface chromatographic column produced peak widths for insulin that were significantly narrower than traditional C18 columns when using formic acid as mobile phase. Mass spectral fragments m/z >700 provided greater specificity, significantly reducing endogenous background. Detection limits in human plasma were 0.2 ng/ml for insulin glargine, glulisine and detemir, and 0.5 ng/ml for insulin aspart. Average accuracy for standard curve and QC samples was 93.4%. CONCLUSION: A simple SPE LC-MS analysis was developed for direct, simultaneous quantification of insulin glargine, detemir, aspart and glulisine.

Challenges in developing an ultra-sensitive bioanalytical method for ethinylestradiol in human plasma.

BACKGROUND: Ethinylestradiol (EE) is the active component in most birth control products. It is especially difficult to analyze due to the presence of many closely related endogenous steroids. Endogenous components can coelute with EE making selective extraction and chromatographic separation challenging. Current MS systems are more sensitive to background, contamination and the overall cleanliness of samples and solvents, placing additional emphasis on sample preparation methodology. METHOD: UPLC was combined with a sensitive triple quadrupole MS and a three-step sample preparation method to highlight and resolve method development challenges. RESULTS: EE was adequately resolved using an unendcapped high-strength silica C(18) column. The average matrix factor in six sources of plasma was 1.14 with a %CV of 4.48. Standard curves were linear with 1/x weighting and r(2) value of 0.999 over three orders of magnitude. Average accuracy for standard curves and quality control samples was 96%. LOD of 0.001 ng/ml was achieved.

Hydrophilic interaction chromatography (HILIC) for LC-MS/MS analysis of monoamine neurotransmitters.

BACKGROUND: Hydrophilic interaction chromatography (HILIC) is becoming an increasingly popular alternative to traditional reversed-phase chromatography for the analysis of polar compounds. The ability to retain the most polar compounds in HILIC makes it attractive for the analysis of certain large groups of compounds, such as monoamines, which are inherently very polar. RESULTS: This paper details the development of a HILIC LC-MS/MS method for the analysis of monoamine neurotransmitters. The emphasis is on method development; in particular, the factors influencing sensitivity, peak shape and resolution. Mobile-phase ionic strength, temperature and stationary phase functionality are shown to be key parameters for the successful development of HILIC methods. CONCLUSION: HILIC is shown to be an appropriate and suitable method for the analysis of monoamine neurotransmitters and an attractive alternative to reversed-phase analysis. The most polar analytes, which are essentially unretained by reversed-phase chromatography, demonstrate superior retention and resolution when analyzed by HILIC.

Influence of stationary phase chemistry and mobile-phase composition on retention, selectivity, and MS response in hydrophilic interaction chromatography.

A comprehensive retention and selectivity characterization of several hydrophilic interaction chromatography (HILIC) stationary phases was performed with 28 test probes in order to study the influence of particle type, surface chemistry, and mobile-phase pH on chromatographic retention, selectivity, and MS response. Selectivity differences were compared for columns operated at both low and high pH, while ESI-MS was used to study the effects of mobile-phase pH on signal response. Additionally, acetone was explored as a potential alternative to ACN as the weak HILIC solvent. Moderate differences in selectivity were observed on the same column operated at different pH, mostly due to acidic compounds. In addition, the MS response increased when a high pH mobile phase was used, particularly for analytes that were ionized with negative ESI-MS. Even larger selectivity differences were observed for different stationary phases evaluated with the same mobile phase. Acetone was not a suitable replacement for ACN in routine HILIC separations due to differences in selectivity and MS response. Finally, the data from this study were used to establish guidelines for rapid HILIC method development of polar compounds, which is demonstrated with a mixture of histidine dipeptides and organophosphonate nerve agent metabolites.

Simultaneous analysis of morphine-related compounds in plasma using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-mass spectrometry.

A bioanalytical method using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the analysis of morphine, morphine-3beta-glucuronide, morphine-6beta-glucuronide, 6-acetylmorphine, morphine N-oxide, and 10-hydroxymorphine in porcine plasma. All six compounds, along with four deuterated internal standards, were simultaneously extracted using mixed-mode strong cation exchange SPE in a 96-well microElution plate format. Due to analyte instability, a neutralizing solvent was used during the elution step to minimize degradation of 6-acetylmorphine. Separation was subsequently performed in 8 minutes on a 2.1 x 100 mm, 1.8 microm C(18 )column designed for retention of extremely polar compounds using a formic acid and methanol gradient. Analytes were detected by positive electrospray ionization in multiple reaction monitoring mode using a fast-scanning triple quadrupole mass spectrometer. Recovery was 73-123% depending on the analyte, and inter-day variability was less than 6%. Linearity was determined in porcine plasma by spiking the analytes prior to SPE. Correlation coefficients were >or= 0.998, and% deviation from the actual concentrations was less than 15%. The lower limit of quantitation (LLOQ) for all compounds was between 0.1 and 0.25 ng/mL.

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-microm porous particles.

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-microm particle columns using both a conventional HPLC system as well as a UPLC system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-microm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-microm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.

Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function.

An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 x 4.1-mm, 10 microm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01-10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5-1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.

Electrospray ionization mass spectrometric analysis of nucleic acids using high-throughput on-line desalting.

A rapid on-line desalting method utilizing ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was employed in tandem with negative electrospray ionization mass spectrometry (ESI-MS) for the routine analysis of nucleic acids. Desalting was performed on a short 10 x 2.1 mm guard column packed with 3.5 microm C(18) sorbent. The HPLC system was connected in-line to an orthogonal ESI-TOF mass spectrometer via a six-port, two-position switching valve, allowing desalting followed by mass analysis of nucleic acids. Duty cycle times for the method were as low as 1.5 min per sample. This allowed for the analysis of approximately 950 samples per 24-h time period, which is suitable for medium- to high-throughput applications. Average mass accuracy was determined to be 80 ppm for oligonucleotides up to 110 mer in length with external calibration. The method was utilized for synthetic oligonucleotide quality control and analysis of DNA genotyping fragments.

Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was utilized in tandem with negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0 x 50 mm column packed with porous C(18) sorbent with a particle size of 2.5 microm and an average pore diameter of 140 A. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an average mass accuracy of 125 ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.

Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography.

Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50x4.6-mm column packed with porous, 2.5 micrometer C(18) sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC-MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy3) dyes, as well as dually-labeled TaqMan probes. Purification of a 0.1-micromole oligonucleotide synthesis in a single injection was demonstrated.

Characterization of therapeutic oligonucleotides using liquid chromatography with on-line mass spectrometry detection.

A method for the analysis and characterization of therapeutic and diagnostic oligonucleotides has been developed using a combination of liquid chromatography and mass spectrometry (LC-MS). The optimized ion-pairing buffers permit a highly efficient separation of native and chemically modified antisense oligonucleotides (AS-ODNs) from their metabolites or failure synthetic products. The mobile phases were MS compatible, allowing for direct and sensitive analysis of components eluting from the column. The method was applied for the quantitation and characterization of AS-ODNs, including phosphorothioates and 2'-O-methyl-modified phosphorothioates. Tandem LC-MS analysis confirmed the identity of the oligonucleotide metabolites, failure products, the presence of protection groups not removed after synthesis, and the extent of depurination or phosphorothioate backbone oxidation.

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.