Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip‐based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C₁₈ LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C₁₈ chips. On the other hand, hydrophobic glycopeptides were better retained on C₁₈ chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four‐protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers

Ion suppression, a matrix effect that affects quantitative mass spectrometry, is one of the main problems encountered in liquid chromatography/tandem mass spectrometry. Two different clean-up steps for the multi-residue analysis of beta-agonists in urine were evaluated with respect to minimisation of ion suppression, namely, a mixed-phase solid phase extraction (SPE) column, i.e., clean screen Dau (CSD), and a molecular imprinted polymer (MIP) SPE column. Ion suppression experiments revealed that CSD sample clean-up can lead to false negative results for some beta-agonists, and that clean-up using MIP columns is more selective for beta-agonists than the use of CSD columns.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

High-throughput liquid chromatography ultraviolet/mass spectrometric analysis of combinatorial libraries using an eight-channel multiplexed electrospray time-of-flight mass spectrometer

We report a high-throughput liquid chromatography/mass spectrometry (LC/MS) protocol for analyzing large combinatorial libraries using an eight-channel parallel LC/UV/MS (MUX-LCT) system. System configuration, linear response range in UV absorbance, LC column selection, and flow rate were optimized for 24 h/7 day unattended operations. Combinatorial libraries were analyzed on this system at a rate of 3200 compounds per day for a 3.5 min cycle time per injection. This parallel system is compared with a single-channel system in terms of performance and operation.     

Bioanalytical applications of 'fast chromatography' to high-throughput liquid chromatography/tandem mass spectrometric quantitation

A fast chromatographic separation approach that enables rapid method development for high-throughput sample quantification is discussed. This approach has been used to analyze samples from various biological matrices. Data are presented from in vivo pharmacokinetic studies (plasma) and in vitro drug metabolism and transport studies (hepatic microsomes, hepatocytes, and Caco-2 cells).     

Ultrafast liquid chromatography/tandem mass spectrometry bioanalysis of polar analytes using packed silica columns

Ultrafast liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalysis was demonstrated with the use of packed silica columns operated under elevated flow rates. A special effort has been made to achieve ultrafast analysis without sacrificing chromatographic resolution. Two multiple analyte/metabolites assays, (1) morphine/morphine-6-glucuronide(M6G)/morphine-3-glucuronide(M3G) and (2) midazolam/1'-hydroxymidazolam/4-hydroxymidazolam, were used to demonstrate the speed, sensitivity, peak shape and separation of the ultrafast methods utilizing silica columns. In both methods adequate chromatographic separation was a necessity because quantitation results would be otherwise compromised due to cross interference between different selected reaction monitoring (SRM) transitions. Baseline resolutions between morphine, M6G and M3G in human plasma extracts were achieved within 30 s on a 50 x 3 mm Betasil silica column operated at 4 mL/min of isocratic acetonitrile/water mobile phase. The total injection-to-injection cycle time was 48 s with a simple, single-autosampler/single-column setup, when a Shimadzu SIL-HT autosampler was used. Baseline resolution between 1'-hydroxymidazolam and 4-hydroxymidalolam in monkey plasma extracts was achieved within 33 s using similar conditions. Due to the absence of carry-over in this case, no rinsing of the injection needle was necessary, resulting in a cycle time of only 39 s/sample. These ultrafast methods were successfully used to analyze extracted biological samples and proved to be reproducible, reliable and generated equivalent pharmaco-kinetic (PK) results to those obtained by regular flow LC/MS/MS analysis to support discovery PK studies.     

Aldehyde analysis by high performance liquid chromatography/tandem mass spectrometry

This paper describes the development of a high performance liquid chromatography/tandem mass spectrometric (MS/MS) procedure for the specific qualitative and quantitative analysis of lipid aldehydes in biological matrices. A derivatisation method, which results in molecules that exhibit a common product ion on MS/MS, permits informative precursor ion scans, at high sensitivity. This has been applied to the examination of plasma in order to examine the production of aldehydes consequent on in vitro lipid oxidation. Quantitative analysis of target molecules using multiple reaction monitoring has been developed to permit quantitation in the same matrices.     

High-throughput chiral liquid chromatography/tandem mass spectrometry

Chiral liquid chromatography is a well-established area of bioanalytical chemistry and is often used during the processes of drug discovery and development. The development and use of a chiral drug require the understanding of the pharmacokinetic characteristics of each of the enantiomers, including potential differences in their absorption, distribution, metabolism, and excretion. Chromatographic techniques coupled to atmospheric pressure ionization-tandem mass spectrometry have shown potential as sensitive and robust tools in the quantitative and qualitative determination of enantiomers in biologic fluids and tissue extracts. However, development of a chiral liquid chromatography method requires time-consuming procedures that are devised empirically. Clearly, there is an incentive to design chromatographic approaches that are easy to use, compatible with mass spectrometry ionization interface conditions, exhibit relatively short run times without compromising sensitivity, and offer a broad analyte specificity. For these reasons, the present paper explores the feasibility of the bonded macrocyclic glycopeptide phases (teicoplanin and vancomycin) for analysis by chiral liquid chromatography/tandem mass spectrometry. Ritalinic acid, pindolol, fluoxetine, oxazepam, propranolol, terbutaline, metoprolol, and nicardipine were tested in this study. Furthermore, an example of a simultaneous chiral LC/MS/MS detection (chromatographic run time approximately 10 min) of four pharmaceutical products resulting in baseline resolutions of all four pairs of enantiomers is presented. Methanol, an MS-compatible mobile phase, was utilized in all the experiments.     

Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection

Four cation-exchange materials, possessing propanesulfonic acid ligands, for use in capillary electrochromatography were prepared from different commercially available 5-microm bare-silica particles ranging from 80 to 800 A in pore size. The performance of the materials was investigated at different compositions of the mobile phase (pH, ionic strength, and acetonitrile content) using tricyclic antidepressants and related quaternary ammonium analogues as test analytes. The wide-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave, as could be expected, the best selectivity.     

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.     

液相色谱-串联质谱法测定蜂蜜和鱼肉中烟曲霉素残留量

提出了液相色谱串联质谱测定蜂蜜和鱼肉中烟曲霉素残留量的分析方法。蜂蜜样品和鱼肉样品分别用磷酸盐缓冲液和乙腈提取,经Oasis MAX固相萃取柱净化后,在Kinetex C18(2.6μm×2.1 mm×100 mm)反相色谱柱上以乙腈-0.1%甲酸酸水溶液(70+30)为流动相色谱分离,采用电喷雾正离子扫描、多反应监测模式(ESI+-MRM)进行质谱检测,外标法定量。方法的测定下限(10S/N)蜂蜜为2μg/kg,鱼肉为5μg/kg。在2～100μg/L的浓度范围内呈良好线性,线性相关系数>0.9997。方法在3个水平的添加回收率在75.4%～79.6%之间,相对标准偏差在6.4%～8.4%之间。方法适用于蜂产品和鱼肉中烟曲霉素残留的定量及确证检测。     

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of navitoclax

The Bcl-2 family small molecule inhibitor navitoclax is being clinically evaluated to treat multiple cancers including lymphoid malignancies and small cell lung cancer. A sensitive and reliable method was developed to quantitate navitoclax in human plasma using liquid chromatography with tandem mass spectrometry with which to perform detailed pharmacokinetic studies. Sample preparation involved protein precipitation using acetonitrile. Separation of navitoclax and the internal standard, navitoclax-d8, was achieved with a Waters Acquity UPLC BEH C₁₈ column using isocratic flow over a 3 min total analytical run time. A SCIEX 4500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of navitoclax. The assay range was 5–5,000 ng/ml and proved to be accurate (89.5–104.9%) and precise (CV ≤ 11%). Long-term frozen plasma stability for navitoclax at −70°C was at least 43 months. The method was applied for the measurement of total plasma concentration of navitoclax in a patient receiving a 250 mg daily oral dose.     

Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock tissues

Ractopamine HCl is a beta-adrenergic agonist (beta-agonist) recently approved by the U.S. Food and Drug Administration, but not other governmental agencies, for use in finishing swine. For these reasons, it was important to develop and validate mass spectrometric methods for the detection and confirmation of ractopamine residues in livestock marker tissues. Incurred tissues in cattle, sheep, turkeys, and ducks were generated during 7-day ractopamine feeding (20 ppm in diets) periods. Disposition of ractopamine residues in liver and pigmented retinal epithelium was determined in animals slaughtered with withdrawal periods of 0, 3, and 7 days. Ractopamine residues, purified using solid-phase extraction, were measured using liquid chromatography (LC) and electrospray with detection by tandem mass spectrometry (MS/MS) in the multiple reaction-monitoring (MRM) mode. Total ractopamine residues (parent ractopamine + hydrolyzed conjugates) in liver were detected in all species on withdrawal day 0 (2-97 ppb) and were greatly diminished in all species by withdrawal day 7 (<1 ppb). Bovine and ovine retina had lower levels of ractopamine (0.5-3 ppb) than liver, and occular residues increased with withdrawal time, suggesting redistribution into this tissue. Lower limits of quantification were found to be approximately 0.1 ppb in liver and retina. Incurred ractopamine residues were confirmed by the precise and accurate agreement of MRM intensity ratios of diagnostic fragment ions (m/z 284, 164, and 121) from the protonated molecule between ractopamine residues in incurred samples and an authentic ractopamine standard. The limits of confirmation in liver and retina using recognized acceptance criteria were below 1 ppb. The high sensitivity and specificity for measurement and confirmation of ractopamine residues suggests this method will be applicable for regulatory residue surveillance programs.     

Importance of using highly pure internal standards for successful liquid chromatography/tandem mass spectrometric bioanalytical assays

Internal standards (IS) with similar physicochemical properties to the analyte provide multiple advantages in liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalytical methods such as: reduction of the analysis run time, improvement in the intra‐injection reproducibility, impact reduction of matrix and ionization effects. However, it is important to evaluate the purity of the IS prior to their use. Indeed, a minor impurity in the IS may lead to an important issue during bioanalytical method development. Stable labelled internal standards are usually appropriate IS for bioanalysis. The use of oxycodone‐D3, ursodiol‐D5 and atovaquone‐D4 as internal standards in three different bioanalytical methods was evaluated. During oxycodone, oxymorphone and noroxycodone simultaneous quantification method development, oxymorphone was identified as a contaminant in oxycodone‐D3. Since the limit of quantification for oxymorphone was very low (10 pg/mL), the presence of an even low percentage of oxymorphone in oxycodone‐D3 leads to the change of the stable labelled IS for an analogue, ethylmorphine. 23‐Nordeoxycholic acid was preferred to ursodiol‐D5 as internal standard for the ursodiol, tauroursodiol and glycoursodiol simultaneous quantification method. Indeed, more than 7% of ursodiol was identified in the ursodiol‐D5 which could not be bypassed by decreasing the IS concentration without compromising the linearity. An atovaquone‐D4 reference standard revealed the non‐negligible presence of atovaquone‐D5 to atovaquone‐D8 that has a large impact on the method validation. Therefore, atovaquone‐D4 was sent for recertification since its isotopic purity was found to be much less than the isotopic purity mentioned on its certificate of analysis. Consequently, during bioanalytical method development, the purity of the IS should be scrutinized. Copyright © 2009 John Wiley & Sons, Ltd.     

An ion-pairing liquid chromatography/tandem mass spectrometric method for the determination of ethephon residues in vegetables

A rapid, sensitive and selective method has been developed for the direct determination of ethephon residues in vegetables (apple, cherry and tomato). Given the anionic character of ethephon, the use of ion-pairing liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS, triple quadrupole) allowed its direct determination in these matrices avoiding a derivatisation step and favouring the automation of the method. Samples were extracted with a mixture of dichloromethane/aqueous formic acid (pH 3) (1:1). Then, tetrabutylammonium acetate (TBA) was added as an ion-pairing reagent, and an aliquot of the aqueous extract was directly injected into the LC/MS/MS system. Quantification was performed with matrix-matched standards prepared from blank sample extracts. MS/MS measurements were made in the selected reaction monitoring (SRM) mode, using the most sensitive transition (m/z 107 > 79) for quantification, and up to four additional transitions for confirmation. Quantitative recoveries were obtained for all matrices (between 83% and 96%) at two concentration levels tested (0.05 and 0.5 mg/kg), with relative standard deviations lower than 9% in all cases. The addition of TBA directly into the sample extract contained in the injection vial was found sufficient to obtain satisfactory LC retention for the analyte. Under these conditions, the absence of ion-pairing reagent in the mobile phase minimised the ionisation suppression for ethephon in the MS source, leading to an increase in the sensitivity of the method and reaching limits of detection of 0.02 mg/kg for all matrices investigated. The acquisition of five specific MS/MS transitions for ethephon allowed the simultaneous and reliable quantification and confirmation of the analyte in the samples.     

An ion-pairing liquid chromatography/tandem mass spectrometric method for the determination of cytarabine in mouse plasma

An ion-pairing high-performance liquid chromatographic (IP-HPLC) system interfaced with an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimal sample preparation was developed for the determination of cytarabine (ara-C), a very hydrophilic anticancer drug, in mouse plasma. A conventional reversed-phase chromatographic column in combination with two ion-pairing reagents was adapted for retention and separation of ara-C from the endogenous interferences in mouse plasma. The effects of the experimental conditions such as the fraction of ion-pairing reagents and organic solvents in the mobile phase on the chromatographic performance and the ionization efficiency of ara-C were investigated. The potential of ionization suppression resulting from the endogenous biological materials on the IP-HPLC/MS/MS method was evaluated using the post-column infusion technique. Furthermore, the feasibility of the proposed IP-HPLC/MS/MS procedure for analysis of ara-C in the mouse plasma was demonstrated by comparison with those obtained by the porous graphite carbon column (PGC) HPLC/MS/MS method.     

Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method

Quantification of orotic acid (uracil-6-carboxylic acid) in urine is an important tool to diagnose some inherited diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. New rapid analytical methods are necessary to provide high-throughput orotic acid analyses. A new analytical method has been developed for the rapid analysis of orotic acid in urine by liquid chromatography coupled with ion spray tandem mass spectrometry (LC/MS/MS). After a sample dilution 1:20, the analysis was performed in the selected reaction monitoring mode in which orotic acid was detected through the transition m/z 155 to 111. The retention time was 3.9 min in a 4.5-min analysis. Daily calibration between 0.5-5.0 micromol/L of orotic acid, corresponding to 10-100 micromol/L in urine before the 1:20 dilution, offered consistent linearity and reproducibility. Interassay coefficient of variance (c.v.) was 4.97% at a mean concentration of 10.99 micromol/L. The sensitivity and specificity of tandem mass spectrometry permitted a high volume of analyses of orotic acid. The sample preparation is simple, inexpensive and not time demanding.     

Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre-cut arteries within pre-defined masses. A high-throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between -4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were < or =5.3% for the quality controls in blood and < or =9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries.     

Post-column infusion study of the 'dosing vehicle effect' in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples

It has become increasingly popular in drug development to conduct discovery pharmacokinetic (PK) studies in order to evaluate important PK parameters of new chemical entities (NCEs) early in the discovery process. In these studies, dosing vehicles are typically employed in high concentrations to dissolve the test compounds in dose formulations. This can pose significant problems for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of incurred samples due to potential signal suppression of the analytes caused by the vehicles. In this paper, model test compounds in rat plasma were analyzed using a generic fast gradient LC/MS/MS method. Commonly used dosing vehicles, including poly(ethylene glycol) 400 (PEG 400), polysorbate 80 (Tween 80), hydroxypropyl beta-cyclodextrin, and N,N-dimethylacetamide, were fortified into rat plasma at 5 mg/mL before extraction. Their effects on the sample analysis results were evaluated by the method of post-column infusion. Results thus obtained indicated that polymeric vehicles such as PEG 400 and Tween 80 caused significant suppression (> 50%, compared with results obtained from plasma samples free from vehicles) to certain analytes, when minimum sample cleanup was used and the analytes happened to co-elute with the vehicles. Effective means to minimize this 'dosing vehicle effect' included better chromatographic separations, better sample cleanup, and alternative ionization methods. Finally, a real-world example is given to illustrate the suppression problem posed by high levels of PEG 400 in sample analysis, and to discuss steps taken in overcoming the problem. A simple but effective means of identifying a 'dosing vehicle effect' is also proposed.     

Data-controlled automation of liquid chromatography/tandem mass spectrometry analysis of peptide mixtures

The structural characterization of proteins and peptides isolated in minute quantities requires the most efficient use of available sample. A mass spectrometer data system was programmed to continuously evaluate incoming liquid chromatography/mass spectrometry data against a user-defined array of information. The resulting conclusions were used to automatically set and modify acquisition parameters in real time to collect collision-induced dissociation spectra for selected ions (tandem mass spectrometry). This approach has provided a mechanism to target specific subsets of masses in a complex mixture and/or to discriminate selectively against masses that are known or not of interest. Masses of contaminants or peptide masses derived from known proteins can be automatically recorded and removed from further consideration for collision-induced dissociation analysis. Once recorded, these “libraries” of masses can be used across multiple analyses. This technique directs the mass spectrometer data system to focus on the analysis of masses significant to the user, even if their signal intensities are well below the intensities of contaminating masses. When combined with a database search program to correlate tandem mass spectra to known protein sequences, the identity of the protein can be established unequivocally by using less than 100 fmol of sample.