Phosphoproteome analysis of human liver tissue by long‐gradient nanoflow LC coupled with multiple stage MS analysis

Reversible protein phosphorylation plays a critical role in liver development and function. Comprehensively cataloging the phosphoproteins and their phosphorylation sites in human liver tissue will facilitate the understanding of physiological and pathological mechanisms of liver. Owing to lacking of efficient approach to fractionate phosphopeptides, nanoflow‐RPLC with long‐gradient elution was applied to reduce the complexity of the phosphopeptides in this study. Two approaches were performed to further improve the coverage of phosphoproteome analysis of human liver tissue. In one approach, ten‐replicated long‐gradient LC‐MS/MS runs were performed to analyze the enriched phosphopeptides, which resulted in the localization of 1080 phosphorylation sites from 495 proteins. In another approach, proteins from liver tissue were first fractionated by SDS‐PAGE and then long‐gradient LC‐MS/MS analysis was performed to analyze the phosphopeptides derived from each fraction, which resulted in the localization of 1786 phosphorylation sites from 911 proteins. The two approaches showed the complementation in phosphoproteome analysis of human liver tissue. Combining the results of the two approaches, identification of 2225 nonredundant phosphorylation sites from 1023 proteins was obtained. The confidence of phosphopeptide identifications was strictly controlled with false discovery rate (FDR)≤1% by a MS²/MS³ target‐decoy database search approach. Among the localized 2225 phosphorylated sites, as many as 70.07% (1559 phosphorylated sites) were also reported by others, which confirmed the high confidence of the sites determined in this study. Considering the data acquired from low accuracy mass spectrometer and processed by a conservative MS²/MS³ target‐decoy approach, the number of localized phosphorylation sites obtained for human liver tissue in this study is quite impressive.     

Phosphoryl group differentiating α‐amino acids from β‐ and γ‐amino acids in prebiotic peptide formation

In recent years β‐amino acids have increased their importance enormously in defining secondary structures of β‐peptides. Interest in β‐amino acids raises the question: Why and how did nature choose α‐amino acids for the central role in life? In this article we present experimental results of MS and ³¹P NMR methods on the chemical behavior of N‐phosphorylated α‐alanine, β‐alanine, and γ‐amino butyric acid in different solvents. N‐Phosphoryl α‐alanine can self‐assemble to N‐phosphopeptides either in water or in organic solvents, while no assembly was observed for β‐ or γ‐amino acids. An intramolecular carboxylic–phosphoric mixed anhydride (IMCPA) is the key structure responsible for their chemical behaviors. Relative energies and solvent effects of three isomers of IMCPA derived from α‐alanine (2a–c), with five‐membered ring, and five isomers of IMCPA derived from β‐alanine (4a–e), with six‐membered ring, were calculated with density functional theory at the B3LYP/6‐31G** level. The lower relative energy (3.2 kcal/mol in water) of 2b and lower energy barrier for its formation (16.7 kcal/mol in water) are responsible for the peptide formation from N‐phosphoryl α‐alanine. Both experimental and theoretical studies indicate that the structural difference among α‐, β‐, and γ‐amino acids can be recognized by formation of IMCPA after N‐phosphorylation. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 232–241, 2003     

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno‐capture of ProGRP in 96‐wells microtiter plates coated with the mAb E146. After immuno‐capture and thorough rinse, trypsin was added for in‐well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC‐MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9–17.5%), accuracy (3.0–6.6%), and linearity (r² = 0.995) in the tested range (0.5–50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC‐MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno‐capture LC‐MS method was superior with respect to sensitivity. This illustrates the large potential of immuno‐capture sample preparation prior to LC‐MS in protein biomarker quantification.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.     

Comparative analysis of salt‐responsive phosphoproteins in maize leaves using Ti4+‐IMAC enrichment and ESI‐Q‐TOF MS

Salinity is one of the most common abiotic stresses encountered by plants. Reversible protein phosphorylation is involved in plant defense processes against salinity stress. Here, we performed global phosphopeptide mapping through enrichment by our synthesized PVA‐phosphate‐Ti⁴⁺ IMAC coupled with subsequent identification by ESI‐Q‐TOF MS. A total of 104 peptide sequences containing 139 phosphorylation sites were determined from 70 phosphoproteins of the control leaves. In contrast, 124 phosphopeptides containing 143 phosphorylated sites from 92 phosphoproteins were identified in salt‐stressed maize leaves. Compared with the control, 47 proteins were phosphorylated, 25 were dephosphorylated, and 45 overlapped. Among the 72 differential phosphoproteins, 35 were known salt stress response proteins and the rest had not been reported in the literature. To dissect the differential phosphorylation, gene ontology annotations were retrieved for the differential phosphoproteins. The results revealed that cell signaling pathway members such as calmodulin and 14‐3‐3 proteins were regulated in response to 24‐h salt stress. Multiple putative salt‐responsive phosphoproteins seem to be involved in the regulation of photosynthesis‐related processes. These results may help to understand the salt‐inducible phosphorylation processes of maize leaves.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of the chemical constituents of the different processed products of Anemarrhena asphodeloides Rhizomes by high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this work, high‐performance liquid chromatography (HPLC) coupled with a hybrid quadrupole time of‐flight mass spectrometry (Q‐TOF‐MS/MS) was used to study chemical compositions of different processed products of Rhizoma Anemarrhenae (RA). A Grace Alltima^TM C₁₈ column (250 × 4.6 mm, 5 µm) was used for separation. Mobile phase consisted of 0.1% formic acid and acetonitrile, using gradient elution. ESI‐MS data was acquired in both positive and negative mode. The experiment was established on the basis of a series of reference substances (two xanthone and seven saponins) to qualitatively identify the chemical compounds of different processed products of RA by MS analysis. There was no difference in the type of chemical constituents between different processed products of RA. A total of 25 compounds were identified, including four xanthones, 21 steroidal saponins and eight pairs of isomers. Copyright © 2015 John Wiley & Sons, Ltd.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

A rapid and sensitive LC‐MS/MS method for evaluation of the absolute oral bioavailability of a novel c‐Met tyrosine kinase inhibitor QBH‐196 in rats

A sensitive, selective and high‐throughput UPLC‐MS/MS method was developed and validated for the determination of a novel c‐Met tyrosine kinase inhibitor, QBH‐196, in rat plasma. QBH‐196 and its analog BH357 (IS) were extracted from rat plasma using a mixture of dichloromethane and N‐hexane (2:3, v/v). The chromatographic separation was carried out on Phenomenex C₁₈ column (50 × 2.1 mm, 2.6 µm particle size) with a gradient mobile phase of methanol (A) and water containing 0.05% formic acid (B) at a flow rate of 0.2 mL/min. The assay was performed by positive electrospray ionization in multiple reaction monitoring mode using transitions of m/z 622.68 → 140.41 for QBH‐196 and m/z 591.19 →126.21 for the IS, respectively. Good linearity was obtained over the concentration range of 8.0–4000 ng/mL (r² > 0.99) for QBH‐196 and the lower limit of quantification was 8.0 ng/mL in rat plasma. Validations of the method, including its sensitivity, extraction recovery, matrix effect, intra‐ and inter‐day precision, accuracy and stability, were all within acceptable limits. The established method was successfully applied to determine absolute oral bioavailability of QBH‐196 in rats for the first time. The mean oral absolute bioavailability of QBH‐196 was found to be about 40.8% and the elimination half‐life was 40.0 ± 13.1 h. This result suggested that QBH‐196 exhibits good oral absorption in vivo, which is very important for the further development of QBH‐196 as a new oral anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Positional isomer differentiation of synthetic cannabinoid JWH‐081 by GC‐MS/MS

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH‐081. Purchased standard compounds of JWH‐081 and its positional isomers were analyzed by gas chromatography‐electron ionization‐mass spectrometry (GC‐EI‐MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near‐identical EI spectra were further subjected to GC‐tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2‐methoxy, 7‐methoxy and 8‐methoxy. The remaining isomers exhibited near‐identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3‐Methoxy and 5‐methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6‐methoxy isomer resembled that of JWH‐081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid determination of parabens in personal care products by stable isotope GC‐MS/MS with dynamic selected reaction monitoring

In this study, a rapid and sensitive analytical method for the determination of methyl‐, ethyl‐, propyl‐, and butyl esters of para‐hydroxy benzoic acid (parabens) in personal care products was developed and fully validated. Test portions were extracted with methanol followed by vortexing, sonication, centrifugation, and filtration without derivatization. The four parabens were quantified by GC‐MS/MS in the electron ionization mode. Four corresponding isotopically labeled parabens were selected as internal standards, which were added at the beginning of the sample preparation and used to correct for recovery and matrix effects. Sensitivity, extraction efficiency, and recovery of the respective analytes were evaluated. The coefficients of determination (r²) were all greater than 0.995 for the four parabens investigated. The recoveries ranged from 97 to 107% at three spiked levels and a one‐time (single) extraction efficiency greater than 97% was obtained. This method has been applied to screen 26 personal care products. This is the first time that a unique GC‐MS/MS method with dynamic selected reaction monitoring and confirmation of analytes has been used to determine these parabens in cosmetic personal care products.     

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.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Distinguishing two isomeric mephedrone substitutes with selective reagent ionisation mass spectrometry (SRI‐MS)

The isomers 4‐methylethcathinone and N‐ethylbuphedrone are substitutes for the recently banned drug mephedrone. We find that with conventional proton transfer reaction mass spectrometry (PTR‐MS), it is not possible to distinguish between these two isomers, because essentially for both substances, only the protonated molecules are observed at a mass‐to‐charge ratio of 192 (C₁₂H₁₈NO⁺). However, when utilising an advanced PTR‐MS instrument that allows us to switch the reagent ions (selective reagent ionisation) from H₃O⁺ (which is commonly used in PTR‐MS) to NO⁺, O₂⁺ and Kr⁺, characteristic product (fragment) ions are detected: C₄H₁₀N⁺ (72 Da) for 4‐methylethcathinone and C₅H₁₂N⁺ (86 Da) for N‐ethylbuphedrone; thus, selective reagent ionisation MS proves to be a powerful tool for fast detection and identification of these compounds. Copyright © 2013 John Wiley & Sons, Ltd.     

Reactivity of Tyr–Leu and Leu–Tyr dipeptides: identification of oxidation products by liquid chromatography–tandem mass spectrometry

The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side‐chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine–leucine (YL) and leucine–tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe²⁺/H₂O₂). Through mass spectrometry (MS), high‐performance liquid chromatography (HPLC‐MS) and electrospray tandem mass spectrometry (HPLC‐MSⁿ) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C‐terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL–YL and LY–LY) and other products as a result of cross‐linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C‐terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

An HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]^– for monosulphonic acids, [M–xH]^x–, and sodiated adducts [M–(x + y)H + yNa]^x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.