Correlation between compensation voltage (CV) and the m/z ratio of singly-charged ions was elucidated. The experimental data for various alkylammonium homologues and various pharmaceutical compounds were used to construct empirical calibration curves that were fit using commercial regression analysis software packages. The best fit equations were applied to calculate the CV differences (ΔCV) in pure N(2) and N(2)/He 50/50 carrier gasses and CV values for a variety of compounds using only m/z values. The calculated values were in good agreement with experimental data and ΔCV values exhibited a very strong correlation with m/z. Application of these empirical calculations may provide a powerful CV prediction tool for researchers using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the value of FAIMS as an analytical method.

Background: The determination of pharmacokinetic parameters requires accurate and reliable bioanalytical methods. Even using highly selective MS/MS, interferences can occur. This paper describes the source of some of these interferences with an example discussed involving the problem of a ketamine interference in a plasma assay. Results: The introduction of field asymmetric waveform ion mobility spectrometry (FAIMS) removed the interference, enhanced signal-to-background and met GLP acceptance criteria. Relative to the non-FAIMS method, assay calibration characteristics were improved. The FAIMS source gave optimal performance following the introduction of a split in order to reduce the inlet flow to approximately 0.4 ml/min. Conclusion: The introduction of ion-mobility separation into a bioanalytical LC-MS/MS method can remove unexpected isobaric interferences without the need to redevelop the chromatography.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an ion-filtering technique recently adapted for use with liquid chromatography/mass spectrometry (LC/MS) to remove interferences during analysis of complex matrices. This is the first systematic study of a series of singly charged tetraalkylammonium ions by FAIMS-MS. The compensation voltage (CV) is the DC offset of the waveform which permits the ion to emerge from FAIMS and it was determined for each member of the series under various conditions. The electrospray ionization conditions explored included spray voltage, vaporizer temperature, and sheath and auxiliary gas pressure. The FAIMS conditions explored included carrier gas flow rate, electrode temperature and composition of the carrier gas. Optimum desolvation was achieved using sufficient carrier gas (flow rate > or = 2 L/min) to ensure stable response. Low-mass ions (m/z 100-200) are more susceptible to changes in electrode temperature and gas composition than high mass ions (m/z 200-700). As a result of this study, ions are reliably analyzed using standard FAIMS conditions (dispersion voltage -5000 V, carrier gas flow rate 3 L/min, 50% helium/50%nitrogen, inner electrode temperature 70 degrees C and outer electrode temperature 90 degrees C). Variation of FAIMS conditions may be of great use for the separation of very low mass tetraalkylammonium (TAA) ions from other TAA ions. The FAIMS conditions do not appear to have a major effect on higher mass ions.

BioanalysisVol. 2, No. 5 General Content - EditorialFree AccessAnalytical confidence and insomniaJames KapronJames KapronThermo Fisher Scientific, 2845 Argentia Ave, Unit 4, Mississauga, ON, L5N 8G6, Canada. Published Online:10 May 2010https://doi.org/10.4155/bio.10.5AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail As a young scientist, I remember dreaming of a perfectly specific analysis in which only my compound of interest was being measured. Some of my contemporaries still wake in the night worried about the quality of their data. With any analysis there will always be the risk of interference, but how can you minimize the risk in LC–MS analyses?This article does not address statistical confidence. Rather, it discusses the risk of having an insufficiently selective analysis. It is about having confidence that you are presenting reliable results to your peers. It also addresses those experiments in need of the highest assurance of correct data: sport doping.This article is separated into several parts: definitions, achieving more confidence via selectivity, an analogy to describe field asymmetric-waveform ion mobility spectrometry (FAIMS) separation, some concerns about this technology, the human characteristics that predispose a scientist to success with new technologies and the final section describes the value of keeping interferences out of the mass spectrometer.DefinitionsSpecificity is generally defined as the measurement of the signal produced by the targeted analyte alone [1]. Selectivity is the measurement of the target analyte to the exclusion of most other compounds in the sample. A perfectly specific method may be obtainable but, like the uncertainty principle, if you have a specific method, you will never be able to prove it. Therefore, the best you can hope for is a highly selective method.Sensitivity among bioanalytical scientists has many definitions. There are four types of sensitivity. For triple quadrupole MS users, instrument sensitivity often refers to the response for a given concentration infused. An alternative definition for MS sensitivity is ion-trap sensitivity. Ion-trap instruments are sensitive, but it comes at the price of time and is related to selectivity. When the trap fills predominantly with the ion of interest (selectivity), it will take longer to fill, but the spectrum at the end will demonstrate very high sensitivity (response).Calibration lines also display sensitivity. This definition is less commonly used but still valid. A sensitive calibration line suggests that a small change in the x-dimension (prepared standard concentration) will result in a large change in the y-dimension (response, calculated concentration). In comparison, if the slope is near zero, then the calibration line may be said to be insensitive.The fourth definition is assay sensitivity. It is a very practical definition and refers to the assay being described as 'fit for purpose.' Assay sensitivity really refers to the ability of the entire system (human operators and instrumentation) to present the supervisor with acceptable results within the limits of the analysis. If there are interferences, the assay characteristics change and the assay sensitivity decreases. Even with a very high signal-to-noise ratio, a very selective method may be obtained, but this still does not address the issue of the unresolved chromatographic or MS interferences.Achieving more confidence via selectivityRemoving interferences increases the selectivity of the analysis and, therefore, increases confidence. Confidence can also be increased by tightly controlling experimental errors and then monitored by statistical analysis. Assuming this is completed with high accuracy and precision, what more can a scientist do to further increase confidence? You need more selectivity.There are numerous techniques readily available to increase selectivity. In the case of the triple-quadrupole MS, higher mass resolution is one option. Another is the selection of an alternate transition for quantitation. In both cases, even though instrument sensitivity may be reduced, signal-to-noise may be increased, providing a better overall result. In the case of chromatography, options include ultra-HPLC for increased chromatographic resolution or stationary phase changes to alter the column selectivity. With sharper chromatographic peaks and altered column selectivity there is a greater likelihood of the resolution of unwanted interferences but, unfortunately, simply applying these options does not always mean the selectivity has actually increased. The interferences may still be lurking beneath the analyte.To improve selectivity, many bioanalytical scientists prefer to adjust the sample-preparation process. However, every additional manipulation introduces sample loss and the risk of user error. Some sample preparation must be performed, but overdoing the purification in the condensed phase provides diminishing returns.These standard methods of improving selectivity (i.e., LC, MS and sample preparation) represent conventional methodologies: baby steps within the same well-defined comfort zone. An alternate dimension of selectivity may be obtained by exploiting a different physical attribute of the analyte.Analogy to describe FAIMS separationField asymmetric waveform ion mobility spectrometry works by ion mobility. Consider twins having leapt from an aircraft; both weigh the same, but they have parachutes of different sizes. As they drift through the atmosphere they do not reach the earth at the same time. But now consider two skyscrapers that apply an electric field on either side of the twins. As they fall and before they reach the ground the electric field pushes and pulls the twins to the left and right. The effective distance travelled is much greater via this zig zag pathway than by a simple linear fall. The longer distance allows for more opportunity to separate the twins from each other. In this analogy, one twin might be the target analyte and the other might be the interference: they possess the same m/z but different flow characteristics through the gas.The main reason to use gas-phase atmospheric pressure separation is because it is still comparatively new and yet sufficiently established that the early teething issues have been worked out of the technology. There is still ample room for publication of novel research. The more adventurous and skilled researchers, by developing a FAIMS method, gain a competitive advantage that other, less adept researchers cannot match. For example, in the case of unscrupulous, doped athletes, it would be very satisfying to catch someone cheating at competitive athletics. New selectivity technologies are a great opportunity to extend the detection time post-dose and level the playing field among athletes [2].Concerns about FAIMSHow much of a structural change will cause a significant separation? Cluster interferences typically are separated from small molecules. Drugs and their metabolites frequently emerge together. Doubly and triply charged tryptic peptides emerge in a band together, but are distinctly separated from singly charged ions. Thus, if the interferences are structurally unrelated to the analytes, they are frequently resolved using standard conditions. It is of particular note that because drugs and metabolites commonly emerge together, compensation voltage switching is not required with these multianalyte assays.The modern FAIMS device uses helium and there is some concern about the cost of this noble gas. The most common way of reducing helium consumption is to use it only when the twins have jumped, not while the aircraft is taxiing for take off, reaching stable altitude or when the plane is landing.Human characteristics for successNew technologies are risky, just like jumping from an airplane. Can we predict the personality characteristics that predispose a tendency to success with new selectivity technologies? The first of the two characteristics is ability. Pasteur has suggested that if your mind is prepared to see the connections, if you have the ability to learn and understand, then you are likely to succeed [3].The second characteristic is an emotional predisposition, a genuine desire to learn and an internal drive to accomplish a goal. For example, are you trying to stay ahead of the competition? Are the stakes for success high? This is true in sport doping analysis, where unscrupulous chemists create new compounds that mask the anabolic steroids [4]. The good guys believe new selectivity techniques serve the world by keeping honest athletes honest.Do you know a scientist with the personality characteristic of obsession with interferences? Are lawyers trying to work ways around their patents? If extra chromatographic peaks allow them to convince the law courts that your data are not reliable, it may be of value to have fewer – or no other – peaks in the chromatogram.Does the quality assurance group give you a headache about slightly quirky chromatographic peak shapes? In high-stake GLP laboratories, a failed analysis might give no result for critical samples. Another outcome of study inspection by a regulatory agency might force the repetition of the study. In serious cases, the withholding of GLP status for that laboratory due to incorrect pharmacokinetic data may give good reason to pursue increased selectivity.The value of keeping interferences out of the mass spectrometerA final word on what selectivity should provide. Traditionally, LC separates compounds and everything is introduced into the MS. Divert valves were invented so that involatile salts do not interfere with instrument robustness. The goal for other selectivity techniques should be similar to divert valves. Mass spectrometers are still very high-priced instruments, despite the gains of recent years. It will always be a bad idea to let everything into your MS. Do not junk it up with ions that are not of interest. Selectively introduce them and let the power of the combined selectivity techniques allow you to sleep better at night. FAIMS is another tool to give you confidence. Experimentally, you will wake up as if your previous troubles were a nightmare, when you realize the problem is solved.Financial & competing interests disclosureThe author is employed by Thermo Fisher Scientific, a manufacturer of field asymmetric waveform ion mobility spectrometry technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Bibliography1 Persson B-A, Vessman J. Generating selectivity in analytical chemistry to reach the ultimate – specificity. Trends Anal. Chem.17(3),117–119 (1998).Crossref, CAS, Google Scholar2 Guddat S, Thevis M, Kapron J, Thomas A, Schaenzer W. Application of FAIMS to anabolic androgenic steroids in sport drug testing. Drug Test. Analysis1(11–12),545–553 (2009).Crossref, Medline, CAS, Google Scholar3 Pasteur L. Dans les champs de l'observation le hasard ne favorise que les esprits préparés. In : Œuvres de Pasteur (Volume 7). Pasteur Vallery-Radot L. (Ed.). Masson, Paris, France, 131 (1922–1939).Google Scholar4 Rivier L. New trends in doping analysis. Chimia56(3),84–90 (2002).Crossref, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 2, No. 5 Follow us on social media for the latest updates Metrics History Published online 10 May 2010 Published in print May 2010 Information© Future Science LtdFinancial & competing interests disclosureThe author is employed by Thermo Fisher Scientific, a manufacturer of field asymmetric waveform ion mobility spectrometry technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

For the determination of trace level impurities, analytical chemists are confronted with complex mixtures and difficult separations. New technologies such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) have been developed to make their work easier; however, efficient method development and troubleshooting can be quite challenging if little prior knowledge of the factors or their settings is available. We present the results of an investigation performed in order to obtain a better understanding of the FAIMS technology. The influence of eight factors (polarity of dispersion voltage, outer bias voltage, total gas flow rate, composition of the carrier gas (e.g. %He), outer electrode temperature, ratio between the temperatures of the inner and outer electrodes, flow rate and composition of the make-up mobile phase) was assessed. Five types of responses were monitored: value of the compensation voltage (CV), intensity, width and asymmetry of the compensation voltage peak, and resolution between two peaks. Three types of studies were performed using different test mixtures and various ionisation modes to assess whether the same conclusions could be drawn across these conditions for a number of different types of compounds. To extract the maximum information from as few experiments as possible, a Design of Experiment (DoE) approach was used. The results presented in this work provide detailed information on the factors affecting FAIMS separations and therefore should enable the user to troubleshoot more effectively and to develop efficient methods.

Mass spectrometric identification of anabolic androgenic steroids challenges standard doping-control methods. To reveal a doping offence the presence of prohibited anabolic androgenic steroids at trace levels in the picogram-per-millilitre range must be confirmed as reliable. Human urine samples containing epitrenbolone, metandienone metabolite (17beta -hydroxymethyl-17alpha-methyl-18-norandrost-1,4,13-trien-3-one), stanozolol, 16beta-hydroxystanozolol and 4beta-hydroxystanozolol were analysed using LC-FAIMS-MS/MS. These substances are prohibited in sport according to World Anti-Doping Agency (WADA) regulations. Glucuronides were hydrolysed and prepared by liquid-liquid extraction. Excellent recovery and precision were obtained for all compounds. Linear calibration results for epitrenbolone and metandienone metabolite were obtained and concentration information could be determined in the ranges of reliable response between 750-1200 and 100-600 pg/mL, respectively. Limits of detection were estimated at 25 pg/mL (stanozolol), 50 pg/mL (metandienone metabolite, 16beta-hydroxystanozolol), 100 pg/mL (4beta-hydroxystanozolol) and 500 pg/mL (epitrenbolone). The assay was applied to doping-control samples. For all analytes, LC-FAIMS-MS/MS resulted in excellent interference removal, which effectively extends the post-dose detection time.

The analysis of peptides presents serious challenges for bioanalytical scientists including low total ion current and non-selective fragmentation during tandem mass spectrometry (MS/MS). During method validation of a peptide in rat serum matrix some interferences could not be easily removed and thus prevented accurate and precise measurement. These problems associated with peptide quantitation were resolved by using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). This selectivity-enhancing technique filters out matrix interferences, and the resulting pseudo-selected reaction monitoring (pseudo-SRM) chromatograms were nearly free from interferences. Control blank matrix samples contained an acceptable level of interference (only 7% signal as compared to the lower level of quantitation). Chromatographic peaks were easily, accurately and precisely integrated resulting in a validated liquid chromatography (LC)/FAIMS-MS/MS method for the analysis of a peptide drug in rat serum according to United States Food and Drug Administration (US FDA) bioanalytical guidelines. These results confirm that new selectivity-enhancing technologies aid the pharmaceutical industry in reliably producing acceptable pharmacokinetic data.

Goal: To improve the accuracy and precision of an LC-MS-MS assay by utilizing high-Field asymmetric waveform ion mobility spectrometry (FAIMS) and highly selective reaction monitoring (H-SRM).

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.

The effect of metabolite interference during liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of an amine drug was investigated using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). The selected reaction monitoring (SRM) transition used for the drug exhibited an interference due to in-source conversion of the N-oxide metabolite to generate an ion isobaric with the drug. The on-line FAIMS device removed the metabolite interference before entrance to the mass spectrometer. FAIMS was used to demonstrate the relative accuracy and precision of drug analysis even in the presence of a co-eluting metabolite that may undergo in-source conversion. Copyright (c) 2005 John Wiley & Sons, Ltd.

A high throughput assay for SCH 211803, an M2 muscarinic receptor antagonist in human plasma using nanoelectrospray infusion tandem mass spectrometry is described. Sample processing consisted of protein precipitation followed by solid phase extraction using octadecasilyl resin-filled pipette tips on a liquid handling robotic system. The sample extracts were infused directly to the mass spectrometer using a nanoelectrospray interface in a silicon chip format. SCH 211803 was quantified in plasma over the concentration range of 1-1000 ng/mL. In comparison with a liquid chromatography-tandem mass spectrometry assay, the nanoelectrospray method has comparable accuracy, precision and limit of quantitation, with a nine-fold improvement in sample throughput. Using the nanoelectrospray assay, ion suppression was evaluated and found to be 15%. This represented a four-fold reduction in matrix suppression when compared to a conventional electrospray source operating in the flow injection analysis mode at a flow rate common for LC-MS/MS analysis.

An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.

A new device is described for sample introduction into the mass spectrometer. It consists of a robotic platform for sample handling based on 96-well plates that delivers samples into a chip-based array of micro-fabricated nanoelectrospray nozzles. The system is designed for laboratories that need unattended bioanalysis, such as pharmaceutical laboratories involved in the determination of small-molecule drugs in biological samples.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Muller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state F-19-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix‐assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 + 250 and 63,507 + 200). Mass spectrometry also revealed molecular heterogeneity associated with both the α and β subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17‐27% carbohydrate by weight, the α subunit contains 0‐30% carbohydrate and the β subunit contains 27‐30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N‐linked glycosylation sites were identified, three in the α subunit (α4N, αy81N, α123N) and three in the β subunit (β64N, β127N, and β147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialo‐biantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site β64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site β147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennay with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated α and β subunits, including all Ser‐ and Thr‐containing peptides. No evidence was found for the presence of O‐linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure‐function relationships and the role clusterin glycosylation plays in physiological function.