Highly sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by solid‐phase extraction and liquid chromatography‐tandem mass spectrometry

Using bamboo‐activated charcoal as SPE adsorbent, a novel SPE method was developed for the sensitive determination of tetrabromobisphenol A and bisphenol A in environmental water samples by rapid‐resolution LC‐ESI‐MS/MS. Important parameters influencing extraction efficiency, including type of eluent, eluent volume, sample pH, volume and flow rate, were investigated and optimized. Under the optimal extraction conditions (eluent: 8 mL methanol, pH: 7; flow rate: 4 mL/min; sample volume: 100 mL), low LODs (0.01–0.02 ng/mL), good repeatability (6.2–8.3%) and wide linearity range (0.10–10 ng/mL) were obtained. Satisfied results were achieved when the proposed method was applied to determine the two target compounds in real‐world environmental water samples with spiked recoveries over the range of 80.5–119.8%. All these facts indicate that trace determination of tetrabromobisphenol A and bisphenol A in real‐world environmental water samples can be realized by bamboo‐activated charcoal SPE‐rapid resolution‐LC‐ESI‐MS/MS.     

Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase,strong cation exchange,weak anion exchange,and fused‐core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C₁₈ at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C₁₈ at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Unveiling the rat urinary proteome with three complementary proteomics approaches

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS‐PAGE separation, followed by LC‐ESI‐MS/MS; 2DE, followed by MALDI‐TOF‐TOF and 2D‐liquid chromatography‐chromatofocusing, followed by LC‐ESI‐Q‐TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases.     

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies

We have evaluated CZE‐ESI‐MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5‐point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE‐MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ～70‐min separation window (～90‐min total analysis duration) and ～300‐peak capacity. We also analyzed the sample using ultra‐performance LC‐MS/MS. CZE‐MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low‐level spiked proteins. Both methods detected all proteins spiked at ～100 ppm level with respect to the antibody.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

Evaluation of different two‐dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large‐scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide‐level fractionation, the first dimension of separation was performed with high‐pH reversed‐phase chromatography (pH‐RP) and strong cation exchange chromatography (SCX). Regarding protein‐level fractionation, C₈ protein reversed‐phase (C₈‐RP Prot) and high‐recovery protein reversed‐phase (hr‐RP Prot) were used instead. The second dimension consisted of a reversed‐phase nano‐HPLC on‐Chip coupled to an electrospray ionization quadrupole time‐of‐flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide‐level pH‐RP and the hr‐RP Prot protein‐level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide‐level SCX, with 509 proteins identified, was the worst method. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive determination of Schisandrin and Schisandrin B in plasma of rats after administration of Wurenchun (Fructus Schisandrae Chinensis Extracts) preparations by LC‐ESI‐MS/MS

A sensitive and specific method based on liquid chromatography‐tandem mass spectrometry using electrospray ionization (LC‐ESI‐MS/MS) has been developed for the determination of Schisandrin and Schisandrin B in rat plasma. A 100 μL plasma sample was extracted by methyl tert‐butyl ether after spiking the samples with nimodipine (internal standard) and performed on an XTerra®MS‐C₁₈ column (150 mm × 2.1 mm, 3.5 μm) with the mobile phase of acetonitrile–water–formic acid (80:20:0.2, v/v) at a flow rate of 0.2 mL/min in a run time of 8.5 min. The lower limit of quantification of the method was 40 ng/mL for Schisandrin and 20 ng/mL for Schisandrin B. The method showed reproducibility with intra‐day and inter‐day precision of less than 13.8% RSD, as well as accuracy, with inter‐ and intra‐assay accuracies between 93.5 and 107.2%. Finally, the LC‐ESI‐MS/MS method was successfully applied to study the pharmacokinetics of Schisandrin and Schisandrin B in rats after administration of Wurenchun commercial formulations to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a validated high-throughput LC-ESI-MS method for determination of sirolimus on dried blood spots

A high‐throughput liquid chromatography–electrospray ionization mass spectrometric (LC–ESI‐MS) method for screening of sirolimus on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase LC on a relatively new monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI‐MS. The run time was less than 3 min with a very low backpressure at a flow rate of 0.5 mL/min. The method can analyze more than 100 samples in an 8 h working day, including sample preparation. The assay was linear from 1 to 100 ng/mL. The mean recovery was 92.42%. The mean inter‐day and intra‐day precisions were 1.23 and 1.41%, respectively. The developed method is simple, rapid and useful for clinical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray ionization mass spectrometric detection of low polar compounds by adding NaAuCl4

Liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) has been widely used for various analyses. However, it is difficult to use LC/ESI/MS for the analysis of low polar compounds, such as polycyclic aromatic hydrocarbons. It is well known that AuCl₄^? ion decomposes to AuCl₃ by heating, and AuCl₃ is a strong π‐electrophilic Lewis acid. Low polar compounds (pyrene, benzo[a]pyrene, perylene, benzo[ghi]perylene, dibenzothiophene and p‐dimethoxybenzene) were detected by ESI/MS in the positive ion mode by adding NaAuCl₄. The low polar compound interacts with AuCl₃ formed at the ESI interface, and undergoes electron transfer to AuCl₃. The radical cation of the low polar compound was then detected by MS. In addition, the LC/ESI/MS determination of polycyclic aromatic hydrocarbons by the post‐column addition of NaAuCl₄ was studied. © 2016 The Authors Journal of Mass Spectrometry Published by John Wiley & Sons Ltd     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Development and validation of an LC‐ESI‐MS/MS quantification method for a potential γ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays

Binding assays for the γ‐aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry‐based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC‐ESI‐MS/MS quantification method for DDPM‐1007 {(RS)‐1‐[4,4,4‐Tris(4‐methoxyphenyl)but‐2‐en‐1‐yl]piperidine‐3‐carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C₈ column in combination with a mobile phase composed of 10 mm ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM‐1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM‐1007 [(²H₉)DDPM‐1007] was synthesized and employed as internal standard. This way DDPM‐1007 could be quantified in a range from 100 pm to10 nm in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra‐ and inter‐batch accuracy. Based on this LC‐ESI‐MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM‐1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM‐1007 at GAT3 could be unambiguously detected. Additionally, the established LC‐MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM‐1007, as exemplified for its logD determination. Copyright © 2012 John Wiley & Sons, Ltd.     

One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

The sensitivity of detection of uric acid (H₂U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pK_a1=5.4 of H₂U, protonated unoxidized uric acid [H₂U+H]⁺ (m/z 169) was detected together with the protonated uric acid dimer [2H₂U+H]⁺ (m/z 337). The dimer likely forms by 1e^? oxidation of urate (HU^?) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e^?, 2H⁺ oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pK_a2=9.8 of H₂U, allantoin (Allnt) (MW 158.04), the final 2e^?, 2H⁺ oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]⁺ (m/z 235) and the [2H₂U+H]⁺ dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]⁺ (pK_sp NaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH₄Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.     

LC‐MS of novel narcoepileptics and related substances

Modafinil, adrafinil and their related substances were synthesized and analyzed by RP‐LC with ESI‐MS/MS. The ionization mode, polarity, cone voltage, and chromatographic conditions were evaluated. The optimum LC‐MS conditions to obtain fragment ions indispensable for identification of the structures were described. The bulk drugs purity of modafinil and adrafinil was evaluated on Kromasil C₁₈ column with ACN/0.02 M ammonium acetate as mobile phase in gradient elution mode at 30°C. The method was found to be suitable not only for monitoring the reactions during the process development but also for quality assurance of modafinil and adrafinil.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.