Journal of Chromatography B (v.825, #2)

Preface by M.G. Bartlett (97).

High performance liquid chromatography with atmospheric pressure ionization (API) mass spectrometry has been essential to a large number of quantitative analytical applications for a variety of compounds. Poor detection sensitivity however is a problem observed for a number of analytes because detection sensitivity can be affected by many factors. The two most critical factors are the chemical and physical properties of the analyte and the composition of the mobile phase. In order to address these critical factors which may lead to poor sensitivity, either the structure of the analyte must be modified or the mobile phase composition optimized. The introduction of permanently charged moieties or readily ionized species may dramatically improve the ionization efficiency for electrospray ionization (ESI), and thus the sensitivity of detection. Detection sensitivity may also be enhanced via introduction of moieties with high proton affinity or electron affinity. Mobile phase component modification is an alternative way to enhance sensitivity by changing the form of the analytes in solution thereby improving ionization efficiency. pH adjustment and adduct formation have been commonly used to optimize detection conditions. The sensitivity of detection for analytes in bio-matrices could also be enhanced by decreasing ion-suppression from the matrix through derivatization or mobile phase addition. In this review, we will discuss detection-oriented derivatization as well as the application of mobile phase additives to enhance the sensitivity of detection in liquid chromatograph/atmospheric ionization/mass spectrometry (LC/API/MS), focusing in particular on the applications involving small molecules in bio-matrices.
Keywords: Reviews; Sensitivity improvement; Derivatization; Mobile phase additives;

The study of the effect of mobile phases on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography (HPLC)–electrospray mass spectrometry (ESI-MS) has been the aim of this review. Reversed-phase chromatography (RPLC) is the chromatographic mode most suitable for coupling with ESI-MS since mobile phases containing organic modifiers are used. The analysis of proteins and peptides by RPLC mostly involves the use of trifluoroacetic acid (TFA) as an ion-pairing agent despite its being a strong suppressor of the MS signal. Different studies reporting the effects of using other ion-pairing agents (other perfluorinated acids, acetic acid, formic acid, etc.) and buffers (ammonium acetate, ammonium formate, ammonium bicarbonate, morpholine, etc.) in RPLC–ESI-MS of proteins and peptides did not yield a single strong candidate that could generally replace TFA. The enhancement in sensitivity with other reagents observed in some cases strongly depended on the analyte, the experimental conditions used, and the mass spectrometer and, usually, it did not compensate for the loss in separation resolution related to TFA. The examples of direct coupling of affinity, size-exclusion, or ion-exchange chromatography (IEC) to ESI-MS are very limited because of incompatibilities related to the use of mobile phases containing high salt concentrations. To overcome this problem, an intermediate desalting step is needed. Multidimensional chromatography, microdialysis, and ion-capture modules can be used to couple these chromatographic modes with ESI-MS. Multidimensional chromatography with RPLC as a second dimension has most often been used. Although most examples involve the trap and analysis in the second dimension of a certain part of the first separation, some comprehensive analyses of the entire sample in the second dimension have also appeared.
Keywords: Reversed-phase chromatography; Size-exclusion chromatography; Ion-exchange chromatography; Affinity chromatography; Protein; Peptide; Electrospray mass spectrometry;

Protein glycosylation analysis by liquid chromatography–mass spectrometry by Manfred Wuhrer; André M. Deelder; Cornelis H. Hokke (124-133).
Liquid chromatography (LC)–mass spectrometry (MS) has developed into an invaluable technology for the analysis of protein glycosylation. This review focuses on the recent developments in LC and combinations thereof with MS for this field of research. Recently introduced methods for the structural analysis of released glycans (native or derivatised) as well as glycopeptides, on normal phase, reverse phase and graphitized carbon LC columns with online MS(/MS) will be reviewed. Performed on nano-scale or capillary-scale, these LC–MS methods operate at femtomole sensitivity and support the further integration of glycosylation analysis in proteomics methodology.
Keywords: Glycopeptide; Glycoprotein; Graphitized carbon; Normal phase; Reverse phase; Sensitivity;

Improving LC–MS sensitivity through increases in chromatographic performance: Comparisons of UPLC–ES/MS/MS to HPLC–ES/MS/MS by Mona I. Churchwell; Nathan C. Twaddle; Larry R. Meeker; Daniel R. Doerge (134-143).
Recent technological advances have made available reverse phase chromatographic media with a 1.7 μm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC–MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC–MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.
Keywords: UPLC; Mass spectrometry; β-Agonists; Isoflavones; Tamoxifen; Ephedra alkaloids;

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5–1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.
Keywords: Mass spectrometry; HPLC/MS; Metabolite identification; Ultra-trace identification;

Increase of the LC–MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection by Ming Li; Yazen Alnouti; Rachael Leverence; Honggang Bi; Arkady I. Gusev (152-160).
Large volume injection (LVI) has systematically been studied to improve LC–MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC–MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC–MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 μL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 μL plasma injections.
Keywords: Large volume injection; On-line SPE; Bioanalytical; Sensitivity improvement; LC–MS/MS;

HPLC–MS employing deuterium oxide and common MS-compatible deuterated additives in the mobile phase with electrospray ionization is shown to be a viable approach for the structural elucidation of impurities in pharmaceutically active agents following initial studies with protic mobile phases. This approach incorporates the hydrogen/deuterium (H/D) exchange reaction where deuterium is substituted for hydrogen at labile sites. Some developmental compounds studied include an amide, amine, lipopeptide, indole and methyl sulfone. H/D exchange is rapid and the chromatographic performance using deuterated mobile phases is comparable to protic counterparts.
Keywords: LC–MS; Hydrogen/Deuterium exchange; Structural determination;

Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.
Keywords: Rhodopsin; Opsin; Pigment; Membrane protein; G protein-coupled receptor; Mass spectrometry; HPLC; Protein mapping; Protein identification; Protein sequencing; LTQ;

Protein–inhibitor complexes analyzed by alkaline capillary LC–MS by Stone D.-H. Shi; Michael J. Greig; James E. Solowiej; Brion W. Murray (176-185).
Liquid chromatography–mass spectrometry (LC–MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein–ligand complexes. We have successfully developed LC–MS method for protein molecular weight measurement using small-bore and capillary LC–MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein–ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.
Keywords: LC–MS; Intact protein; Alkaline mobile phase; Negative ion; Reversible covalent binding; Sensitivity;

Trifluoroacetic acid (TFA) is a commonly used additive in HPLC and LC–MS analysis of basic compounds. It is also routinely added to aqueous–organic mobile phases utilized in the hydrophilic interaction chromatography–electrospray tandem mass spectrometry (HILIC–ESI/MS/MS) technique used in our laboratories for bioanalysis. However, TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions. The most common method to overcome this problem involves the post-column addition of a mixture of propionic acid and isopropanol. However the post-column addition setup requires additional pumps and is not desirable for continuous analysis of large amounts of samples. In this paper we present a simple yet very effective means of minimizing the negative effect of TFA in bioanalysis by direct addition of 0.5% acetic acid or 1% propionic acid to mobile phases containing either 0.025 or 0.05% TFA. A factor of two- to five-fold signal enhancement was achieved for eight basic compounds studied. Furthermore, chromatography integrity was maintained even with the addition of acetic acid and propionic acid to existing TFA mobile phases. This method has been successfully applied to the HILIC–ESI/MS/MS high-throughput analysis of extracted biological samples to support pre-clinical and clinical studies.
Keywords: Trifluoroacetic acid; TFA; Electrospray; Liquid chromatography–mass spectrometry; LC–MS; LC–MS/MS; Hydrophilic interaction chromatography; HILIC; Silica column;

Reversed phase ion-pair chromatography (RP-IPC) of seven heterocyclic aromatic amines encompassing quinoline (IQ, MeIQ), quinoxaline (MeIQx), pyridine (PhIP) and carboline derivatives (AαC, Harman, Norharman) was carried out with formate as counter ion in an aqueous eluent with acetonitrile as organic modifier. TSKgel ODS-80TS was used as the stationary phase. With the aim of acquiring a better insight into the mutual influence of ion-pair reagent and the organic modifier upon solute retention, the study was performed by using an experimental design approach able to evidencing the effect of the simultaneous variation of the two factors. A model for the chromatographic behavior of the amines is proposed that includes classical ion-pair mechanism involving formate in the case of MeIQx, PhIP, Harman and Norharman. A competitive ion-exchange mechanism was hypothesized to govern retention of quinoline compounds, whereas electrostatic interactions and hydrogen bond formation with the silanols of the stationary phase were judged to be responsible for the retention of AαC. Further, the chromatographic behavior of the analytes using the formic acid-ammonium formate buffer in the mobile phase was compared with that observed using acetic acid-ammonium acetate buffer. The method based on the use of RP IPC with tandem mass spectrometry when the eluent contained formate buffer at pH 2.8 exhibited higher detectability with respect to that achieved using the acetate buffer.
Keywords: Heterocyclic aromatic amines; Liquid chromatography–mass spectrometry; Ion-pair reagents;

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography–mass spectrometry (LC–MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro C18 column eluted with a mobile phase consisting of hexafluoro-2-propanol and triethylamine in methanol at 50 °C. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC–MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.
Keywords: Pharmacokinetics; Tandem mass spectrometry; Metabolism; Bcl-2 antisense; N-in-one quantification;

A derivatization reagent, 2-hydrazino-1-methylpyridine, was developed for the liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) of oxosteroids. The reagent quantitatively reacted with oxosteroids at 60 °C within 1 h and the resulting derivatives of the mono-oxosteroids provided a 70–1600-fold higher sensitivity compared to intact steroids. However, HMP was unsuitable for di-oxosteroids, such as androstenedione and progesterone. The developed derivatization procedure was applied to the LC–ESI-MS analysis of 5α-dihydrotestosterone in human prostate, and allowed the reproducible quantification of nanogram/gram level of the androgen with a 10-mg sample.
Keywords: Derivatization; LC–ESI-MS; Oxosteroids; Human prostate;

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50–10000 pg/ml and 2.5–500 pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-13C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC–MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm × 4.6 mm, 3 μm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug–drug interaction studies in human subjects.
Keywords: Norethindrone; Ethinyl estradiol; LC–MS/MS; Derivatization; Simultaneous analysis;

A method is presented for improved detection of pseudouridine in nucleoside mixtures based on the specific derivatization with methyl vinyl sulfone followed by analysis by capillary HPLC–mass spectrometry. Reaction conditions were optimized in order to obtain the best yield and specificity. The method was successfully applied to different nucleoside mixtures.
Keywords: Pseudouridine; Modified nucleosides; RNA;

Author Index (239-240).

Keyword Index (241-245).