Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

Hybrid mode of hydrophobic interaction chromatography of monoclonal antibodies and related biomolecules: Influence of elution conditions on chromatographic performance using poly (alkyl aspartamide) silica columns.

A hybrid mode of hydrophobic interaction chromatography (HHIC) is an emerging analytical technique for the separation of biomolecules under non-denaturing conditions that combines elements of conventional hydrophobic interaction and reversed-phase chromatography. This article explores the impact of mobile phase composition such as salt concentration and organic modifier on the separation of therapeutic monoclonal antibodies and related large biomolecules using poly (alkyl aspartamide) silica HIC columns. The initial mobile phase salt concentration had a significant impact on the separation of a mixture of large biomolecules demonstrating that the relationship of elution and salt concentration was more complex than in conventional HIC. In general, the earlier eluting components exhibited greater retention at higher salt concentration as is typical of HIC separations. Conversely, the later eluting components showed greater retention at lower initial salt concentration. This differential is useful for improving the overall separation by widening the elution window for components of a mixture. In addition, no significant unfolding of the proteins was detected by intrinsic fluorescence or electrospray mass spectrometry. The impact of linear velocity and gradient steepness was also evaluated.

Cyclization of N-Terminal Glutamic Acid to pyro-Glutamic Acid Impacts Monoclonal Antibody Charge Heterogeneity Despite Its Appearance as a Neutral Transformation.

Pyroglutamic acid (pyroGlu) is commonly observed at the N-terminus of therapeutic monoclonal antibodies. Notably, the term "pyroGlu" refers to a single product that could originate from the cyclization of either an N-terminal glutamine or an N-terminal glutamic acid. This is an important and easily overlooked distinction that has major implications on the charge variant nature of a pyroGlu relative to its uncyclized form. Cyclization of an N-terminal glutamine for instance clearly produces an acidic variant with a lower isoelectric point owing to the loss of the positively charged N-terminal amine. In this report, we demonstrate that cyclization of an N-terminal glutamic acid on the other hand produces a basic variant with a higher isoelectric point contrary to the typical assumption that the simultaneous loss of the N-terminal amine and the carboxylic acid side-chain would negate the formation of a charge variant. The results of our investigation demonstrate the need to consider the relative strengths of the acidic and basic functional groups which are altered when assessing whether the product will be a charge variant. This study also adds new knowledge and experimental evidence to understand charge heterogeneity in monoclonal antibodies.

Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion ("pulse" proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis-sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.

Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions.

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions.

Improving Mass Spectral Quality of Monoclonal Antibody Middle-Up LC-MS Analysis by Shifting the Protein Charge State Distribution.

Monoclonal antibodies (mAbs) are experiencing accelerated development in the pharmaceutical industry. Utilization of middle-up LC-MS methodology can provide detailed characterization of mAbs via reduction and/or enzymatic cleavage of the mAb into smaller protein fragments. However, under typical LC-MS conditions, these fragments, especially the more heterogeneous heavy chain, can present charge state distributions (CSD) featuring a severe interference in the low mass-to-charge (m/z) region in the mass spectrum, adversely impacting spectral quality of these proteins and ultimately the deconvoluted mass spectrum. Here, we introduce a novel method to characterize protein fragments by partially reducing mAbs and using acidic mobile phases (MPs) with a trace amount of base additive. Gas-phase charge stripping occurs with the basic MP additive, causing the CSD to shift to a higher m/z region resulting in high-quality mass spectra with enhanced resolution of protein charge states. Subsequently, high-quality deconvoluted spectra and accurate mass measurement of the fragments are achieved. This method has been applied to the intact mass measurement of mAbs and antibody drug conjugates.

Enablement of the direct analysis of excipients in monoclonal antibody formulations through the incorporation of a wide pore C18 protein trap with hydrophilic interaction liquid chromatography.

Establishing and maintaining the correct formulation composition is essential for ensuring the stability of biopharmaceutical drug products. A barrier to the routine assessment of excipient concentration is the lack of convenient and robust methods for the direct analysis of solutions containing high protein concentrations. To address this need an HPLC method was developed utilizing a wide-pore C18 guard column to trap proteins in-line with a hydrophilic interaction liquid chromatographic column to separate excipients. This method allows for a simple and direct analysis of excipients such as amino acids, carboxylic acids, simple carbohydrates, and inorganic ions across multiple drug formulations and process streams containing different therapeutic antibodies. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.

Identification of leachables observed in the size exclusion chromatograms of a low concentration product stored in prefilled syringes.

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined analytical methods on a subset of materials intended to be representative of the marketed product. Throughout product development, leachables may occasionally be detected in other methods not specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC-MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR (RPLC-SPE-NMR), as initial RPLC-MS/MS investigations were unsuccessful in elucidating the structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous mixed mode separation mechanism and low concentration nature of the product, which were the main factors contributing to the unlikely detection of the leachables by SEC. We note that our investigations were conducted independently of formal leachables and extractables (L&E) studies and we discuss challenges with designing and conducting such studies in a manner that captures the comprehensive L&E profile of a product.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.

Exploitation of the size-exclusion effect of reversed-phase high performance liquid chromatography for the direct analysis of diethylene triamine pentaacetic acid in therapeutic monoclonal antibody formulations.

Monoclonal antibodies (mAb) are being widely studied for the treatment of cancers and other diseases. The mAb is typically in a solution formulation and administered as an intravenous infusion. Ready-to-use solutions are favored for their clinical convenience but they can potentially suffer from a shorter shelf life due to accelerated rates of some forms of degradation such as oxidation, relative to lyophilized formulations. To improve stability, the chelating agent diethylene triamine pentaacetic acid (DTPA) is often used at very low concentrations in biologics formulations to prevent oxidation induced by metal ions. Because of its low concentration and susceptibility to changes in concentration during stability study or processing, the measurement of DTPA levels during formulation and process development is critical. In response to this need we developed a platform reversed-phase HPLC method that allows for the rapid and direct determination of DPTA concentrations which does not require the prior removal of mAbs in formulation samples. The method exploits the "size exclusion effect" of C18 columns with narrow pore sizes (90-120Å) to elute large mAb at the void volume, enabling direct injections of mAb samples for quantitation of DTPA. The method was found to be suitable for the analysis of DTPA in the range of 2-20µg/mL across multiple drug formulations containing different therapeutic mAb and antibody drug conjugates. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.

Structural characterization of low level degradants in aztreonam injection and an innovative approach to aid HPLC method validation.

Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products. Two of the new unknown Degradants (I and II) were observed in chromatograms from stability samples of aztreonam injection. The third new Degradant (III) was observed during a stability study of the aztreonam active pharmaceutical ingredient. These degradants were structurally characterized. A small amount (ca. 1-3mg) of each degradant was isolated via preparative HPLC for structure elucidation using accurate MS, one and two-dimensional NMR spectroscopy. The small amount of each NMR sample was then reused as a standard for HPLC purity/impurity method validation. Their exact concentrations were determined using quantitative NMR which enabled the execution of the quantitative elements of the HPLC method validation. This innovative approach eliminated the need to isolate or synthesize larger quantities of markers for HPLC/UV method validation, thus saving significant time and reducing costs.

Trace level liquid chromatography tandem mass spectrometry quantification of the mutagenic impurity 2-hydroxypyridine N-oxide as its dansyl derivative.

A derivatization LC-MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC). The derivatization reagent 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) was used in a basic solution to convert HOPO into the corresponding dansyl-derivative. The derivative was separated from different APIs and reagents by liquid chromatography. The detection of the HOPO dansyl-derivative was achieved by mass spectrometry in selected reaction monitoring (SRM) mode. The LC-MS/MS method had a reporting limit of 0.1ng/mL HOPO, which corresponds to 0.1ppm HOPO relative to an API at 1mg/mL, and a linearity range of 0.1-25ng/mL HOPO analyte. Recoveries of HOPO standards spiked into three different API matrices at 0.2, 1.2, and 20ppm levels were all within 90-100%. An SRM-based confirmatory methodology using the ratios of two fragment ions at three CID energies was developed to verify the identity of HOPO when present at ≥0.6ppm. This identity confirmation can be employed to prevent potential false positive detection of mutagenic impurities at trace level. It can be broadly applicable for the confirmation of analytes when the analytes generate at least two major fragments in tandem mass spectrometry experiments.

A novel route to recognizing quaternary ammonium cations using electrospray mass spectrometry.

Characterizing and elucidating structures is a commonplace and necessary activity in the pharmaceutical industry with mass spectrometry and NMR being the primary tools for analysis. Although many functional groups are readily identifiable, quaternary ammonium cations have proven to be difficult to unequivocally identify using these techniques. Due to the lack of an N-H bond, quaternary ammonium groups can only be detected in the (1)H NMR spectra by weak signals generated from long-range (14)N-H coupling, which by themselves are inconclusive evidence of a quaternary ammonium functional group. Due to their low intensity, these signals are frequently not detected. Additionally, ions cannot be differentiated in a mass spectrum as an M(+) or [M + H](+) ion without prior knowledge of the compound's structure. In order to utilize mass spectrometry as a tool for determining this functionality, ion cluster formation of quaternary ammonium cations and non-quaternary amines was studied using electrospray ionization. Several mobile phase modifiers were compared; however, the addition of small amounts of trifluoroacetic acid proved superior in producing characteristic and intense [M +2TFA](-) clusters for compounds containing quaternary ammonium cations when using negative electrospray. By fragmenting this characteristic ion using CID, nearly all compounds studied could be unambiguously identified as containing a quaternary ammonium cation or a non-quaternary amine attributable to the presence (non-quaternary amine) or absence (quaternary ammonium cation) of the resulting [2TFA + H](-) ion in the product spectra. This method of analysis provides a rapid, novel, and reliable technique for indicating the presence of quaternary ammonium cations in order to aid in structural elucidation.

Improving the efficiency of quantitative (1)H NMR: an innovative external standard-internal reference approach.

The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Mitigation of signal suppression caused by the use of trifluoroacetic acid in liquid chromatography mobile phases during liquid chromatography/mass spectrometry analysis via post-column addition of ammonium hydroxide.

A method has been developed to reduce the mass spectrometric ion signal suppression associated with the use of TFA as an additive in LC mobile phases. Through post-column infusion of diluted NH(4)OH solution to LC eluents, the ammonium ion introduced causes the neutral analyte-TFA ion pair to dissociate which consequently releases the protonated analyte as free ions into the gas phase (through regular electrospray ionization mechanisms). An ion signal improvement from 1.2 to 20 times for a variety of compounds had been achieved through the application of this method. The molar ratios of NH(4)OH:TFA which result in a reduction of signal suppression were determined to be between 0.5:1 and 50:1. In addition, it was shown that this NH(4)OH infusion method could reduce the level of doubly-charged species and the product ions formed via in-source collision. The use of diluted NH(4)OH solution is favorable since it is compatible with mass spectrometry analysis, and it is applicable in both positive and negative-ion generation mode.

Separation and detection of bis-maleimide-polyethylene glycol and mono-maleimide-polyethylene glycol by reversed-phase high pressure liquid chromatography.

Monofunctional maleimide polyethylene glycol (mono-mal-PEG) with average molecular weight up to 40 kDa can be used as a raw material for the PEGylation of therapeutic proteins. A possible impurity in this raw material which needs to be controlled is the bisfunctional maleimide-PEG, which has a similar average molecular weight to mono-mal-PEG. Chromatographic separation and detection of low level bis-mal-PEG in mono-mal-PEG presents a major challenge because of the polydispersity of the analytes and the minor difference between the desired mono-mal-PEG and the bis-mal-PEG impurity. In this study, linear mal-PEGs were first derivatized with a specially designed cys-peptide containing a UV chromophore and multiple ionizable sites. Separations were then carried out by reversed-phase HPLC with UV detection at 360 nm. Mono-mal-PEG and bis-mal-PEG were well resolved using a Gemini C18 column with an aqueous-acetonitrile mobile phase. Retention times increased as PEG molecular weight increased from 10 kDa to 40 kDa, while selectivities decreased as PEG molecular weight increased. Results from systematically designed studies for optimization of critical parameters including gradient slope, column temperature, and acidic modifier in the mobile phase led to the selection of the final separation conditions. The developed method conditions were specific, accurate, and sensitive for detecting bis-mal-PEG as an impurity in mono-mal-PEG with limit of quantitation of 0.2% and may be used to assess the quality of mono-mal-PEG as a raw material for protein PEGylation.

Development of a two-step tier-2 dissolution method for blinded overencapsulated erlotinib tablets using UV fiber optic detection.

Measuring dissolution of a comparator drug overencapsulated in a hard gelatin shell is necessary when determining performance of the native and blinded formulations. However, the gelatin in the shell may form cross-links upon storage at stressed conditions, resulting in slow dissolution of the encapsulated drug. The aim of this study was to develop a dissolution approach for a hard-gelatin overencapsulated formulation of a comparator drug, erlotinib, which can overcome cross linking of the capsule shell. In this case, following the USP two-tier dissolution test by simply adding an enzyme did not dissolve the cross-linked capsules because the medium used in the method for erlotinib described in the FDA Dissolution Database contains sodium dodecyl sulfate that inhibits the activity of the enzyme. Changing the method by using different surfactants was not considered acceptable because it is preferable to closely follow the compendial method for the comparator. A two-step tier-2 method was developed as a solution, without significant change to the compendial method conditions. It uses 0.1N HCl + pepsin as the initial medium to help capsule break-up. SDS is added at 15 min after the testing starts to ensure dissolution of the drug. This may be a useful general approach for dealing with cross-linking in over-encapsulated comparators. A UV fiber optic spectrophotometer was used for in situ, real-time detection of the dissolution profile during method development studies. The fast sampling rate available with this type of detection was important in elucidating the events occurring during dissolution and determining the optimal time of the SDS addition.

A combined desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) source for mass spectrometry.

A source that couples the desorption ionization by charge exchange (DICE) and desorption electrospray ionization (DESI) techniques together was demonstrated to broaden the range of compounds that can be analyzed in a single mass spectrometric experiment under ambient conditions. A tee union was used to mix the spray reagents into a partially immiscible blend before this mixture was passed through a conventional electrospray (ES) probe capillary. Using this technique, compounds that are ionized more efficiently by the DICE method and those that are ionized better with the DESI procedure could be analyzed simultaneously. For example, hydroquinone, which is not detected when subjected to DESI-MS in the positive-ion generation mode, or the sodium adduct of guaifenesin, which is not detected when examined by DICE-MS, could both be detected in one experiment when the two techniques were combined. The combined technique was able to generate the molecular ion, proton and metal adduct from the same compound. When coupled to a tandem mass spectrometer, the combined source enabled the generation of product ion spectra from the molecular ion and the [M + H](+) or [M + metal](+) ions of the same compound without the need to physically change the source from DICE to DESI. The ability to record CID spectra of both the molecular ion and adduct ions in a single mass spectrometric experiment adds a new dimension to the array of mass spectrometric methods available for structural studies.