Journal of Chromatography B (v.908, #C)

Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines by Jinmei Fu; Jacob Bongers; Li Tao; Dan Huang; Richard Ludwig; Yunping Huang; Yueming Qian; Jonathan Basch; Joel Goldstein; Ramji Krishnan; Li You; Zheng Jian Li; Reb J. Russell (1-8).
► Alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by LC/MS/MS. ► The sequence variants were confirmed by use of synthetic peptide. ► DNA sequencing of the mater cell bank revealed one variant was caused by mutation at the DNA level.Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8–9.9% and 0.5–0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC–UV with detection at 280 nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC–MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants’ levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.
Keywords: Sequence variant; Monoclonal antibody; High performance liquid chromatography; Tandem mass spectrometry; Peptide mapping;

Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening by Thomas P. Mechtler; Thomas F. Metz; Hannes G. Müller; Katharina Ostermann; Rene Ratschmann; Victor R. De Jesus; Bori Shushan; Joseph M. Di Bussolo; Joseph L. Herman; Kurt R. Herkner; David C. Kasper (9-17).
► We report a short-incubation mass spectrometry-based protocol for lysosomal enzymes. ► Optimized sample handling and online clean-up allowed a 4 h analysis. ► Up to 5 lysosomal enzyme activities are analyzed simultaneously from dried blood spots. ► Method validation was performed under routine clinical laboratory environment.The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within 4 h including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid–liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3 h revealed in intra-day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography–ultra high performance liquid chromatography–tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day.
Keywords: Short-incubation; Lysosomal storage disorders; Multiplex assay; Tandem mass spectrometry; Newborn screening; Turbulent flow chromatography;

Sensitive determination of the peptide AP301 – A motif of TNF-α – From human plasma using HPLC–MS/MS by Daniel Mascher; Werner Tscherwenka; Hermann Mascher; Bernhard Fischer (18-22).
► A new method is described for the determination of AP301 from human plasma. ► A LLOQ of 1 ng/mL using HPLC–MS/MS is reached. ► This provides maximum information of penetration of it from lung into circulation. ► Determination of this compound in a biological matrix has not been published yet.The AP301 peptide mimics the lectin-like domain of TNF-α. The synthetic peptide AP301 (molecular weight 1923.1 amu) is composed of 17 amino acids and contains an intramolecular disulfide bond between the N-terminal and the C-terminal cysteine. AP301 interacts with the endothelial sodium channel (ENaC) and activates pulmonary liquid clearance both in vitro and in animal studies. Currently, AP301 is subject to clinical investigations for the treatment of pulmonary oedema. With HPLC–MS/MS on reversed phase chromatography a determination limit of 1 ng AP301/mL human plasma can be achieved. The MS-ionisation was done with ESI positive. 50 μL of human plasma was mixed with the internal standard (a stable isotope labelled AP301, with a total of 6 carbon 13) in acetonitrile for protein precipitation. After centrifugation a part of the clear supernatant was injected into HPLC–MS/MS. Validation was performed according to FDA-guideline in three batches [U.S. Department of Health and Human Services, Food and Drug Administration (FDA): Guidance for Industry, Bioanalytical Method Validation, May 2001]. By using a 6xC13 isotopically labelled internal standard good precision, accuracy and linearity can be gained. The inter-batch precision (CV) of the quality control samples in human plasma (conc. 2.50/20/240 ng/mL) ranged from 5.54 to 10.15%. The inter-batch accuracy (with reference to the mean value) of the quality control samples in plasma ranged from 96.1% to 99.9%. The analyte was stable in human plasma over three freeze/thaw cycles, or for 4 h at room temperature, or for at least 20 weeks when stored at below −20 °C. This method was used for quantifying AP301 after inhalative application in a phase I-study.
Keywords: HPLC–MS/MS; Sensitive peptide determination; Motif of TNF-α; AP301; Human plasma;

Analysis of coenzyme Q10 in lymphocytes by HPLC–MS/MS by A. Arias; J. García-Villoria; A. Rojo; N. Buján; P. Briones; A. Ribes (23-26).
► The reference range of CoQ10 was narrower when normalized to units of citrate synthase than when normalized to grams of protein. ► Isolated lymphocyte lysates are a reliable matrix for the diagnosis and follow-up of patients with CoQ10 deficiency. ► HPLC–MS/MS is a good procedure to measure CQ10.Coenzyme Q10 (CoQ10) deficiency syndromes are potentially treatable disorders. Skeletal muscle is the most widely accepted tissue for their study, but sampling is an invasive procedure. Cultured skin fibroblasts seem to improve the biochemical diagnosis, but their growth requires a certain period of time. Our aim was to set up a minimally invasive, fast and reliable analytical procedure to measure CoQ10 in lymphocytes, to prevent any delay in diagnosing primary CoQ10 deficiency. HPLC–MS/MS analysis of CoQ10 showed high sensitivity and specificity. The reference range was established in apparently healthy volunteers (n  = 33); the mean of CoQ10 in lymphocytes was 107 nmol/g protein (95% confidence interval: 105–120) and 2.0 nmol/UCS (95% confidence interval: 2.06–2.46). Therefore, the range was narrower when normalized to units of citrate synthase (UCS) than when normalized to grams of protein. The method was linear from 0.01 to 1 μM with a good precision and sensitivity (limit of quantification 0.01 μM). Intra-assay and inter-assay coefficients of variation were lower than 13%. Recovery was higher than 95%. In our hands, lymphocytes seem to be a reliable matrix as they reflect intracellular content of CoQ10. In addition, they can be obtained by a minimally invasive procedure (venipuncture).
Keywords: Coenzyme Q10; Lymphocytes; HPLC–MS/MS;

► We developed a sensitive method for determining β-blockers and β-agonists using LC–MS. ► The method was tested for trace determinations of eleven β-blockers and β-agonists. ► Recovery of 77.20–97.30% and detection limits of 0.11–6.74 pg/ml were obtained. ► Drugs at concentrations of 3.44–234.28 pg/ml were found in Al-Ain and Abu Dhabi WWTPs. ► Results suggest that treatment processes are not enough to degrade these compounds.A highly sensitive method for simultaneous determinations of eleven β-blockers and β-agonists in distilled and waste-waters using liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS–MS) was developed, optimized and validated. The method was used for trace determinations of acebutolol, atenolol, metoprolol, propranolol, timolol, nadolol, labetalol, oxprenolol, pindolol, alprenolol and terbutaline. Oasis MCX and Clean Screen cartridges were used for solid phase extractions and an alkaline mixture of dichloromethane–propanol was used as mobile phase. Matrix effect was reduced by using methanol as a pre-eluant for removing co-extractives on the SPE cartridges and by applying the internal standard method for quantification. Using Oasis MCX-SPE cartridges, developed method gave average recoveries of 77.20–97.30% for drugs spiked at 150.00–500.00 pg/ml. Intra-day precisions gave RSD of 3.367–12.489% while as inter-day precisions gave RSD of 6.425–19.768%. Detection limits of 0.11–6.74 pg/ml and quantification limits of 0.14–22.88 pg/ml were obtained. Signal's suppression in the range of 4.50–24.50% was recorded due to the matrix effect. Drugs spiked in wastewater at 500.00 pg/ml concentrations level and stored at 4 °C for 6 days, showed insignificant degradation. Developed method was successfully applied to the analysis of pharmaceutical residues in effluents wastewaters. Five β-blockers and one β-agonists were detected in Al-Ain and Abu Dhabi wastewaters at average concentrations of 3.44–19.05 pg/ml. Atenolol was detected at higher average concentration ranged in 125.60–234.28 pg/ml. Results obtained suggest that adopted wastewater treatment processes are not enough to degrade these compounds.
Keywords: β-Blockers; β-Agonists; LC–MS–MS; Distilled water; Wastewater;

Pharmacokinetics of protocatechuic acid in mouse and its quantification in human plasma using LC–tandem mass spectrometry by Wei Chen; Dian Wang; Li-shu Wang; Di Bei; Jiang Wang; William A. See; Susan R. Mallery; Gary D. Stoner; Zhongfa Liu (39-44).
► We developed and validated an LC–MS/MS method for the quantification of PCA in plasma. ► This method was used to characterize the pharmacokinetics of PCA in the mouse. ► This method detected PCA in the plasma of patients following oral ingestion of BRB. ► This method provides a translational tool for preclinical and clinical studies of PCA.Protocatechuic acid (PCA), a major microbial-mediated metabolite of anthocyanins, has significant anti-oxidative and anti-carcinogenic activities in vitro and in vivo; however, its pharmacokinetics remains largely unknown. In this report, a sensitive and rapid LC–MS/MS method was developed and validated for the measurement of PCA concentrations in both mouse and human plasma. This method showed a linearity of 1–1000 ng/mL in both mouse and human plasma with a lower limit of quantification of 1 ng/mL. The within-day and between-day coefficient of variation ranged from 1.18 to 11.8% and accuracy from 92 to 110%. The method was applied to characterize the pharmacokinetics of PCA in mice after oral administration of 50 mg/kg PCA. PCA was absorbed rapidly with a half-life of 2.9 min, reached a peak plasma level (C max) of 73.6 μM at 5 min, and remained detectable up to 8 h with the initial elimination half-life of about 3 min and a terminal half-life of 16 min. The area under the plasma concentration–time curve (AUC0→8 h) of PCA was 1456 μM min. The method was capable of detecting low ng/mL quantities of PCA in the plasma of patients with prostate cancer after an oral ingestion of 60 g of black raspberry (BRB) powder. Because PCA is derived from the anthocyanins in BRB, our method provides a useful analytical tool to further investigate the metabolism of anthocyanins, and the pharmacology of PCA in future pre-clinical and clinical studies.
Keywords: Protocatechuic acid; LC–MS/MS; Pharmacokinetics; Plasma;

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin by Aiye Liang; Jay N. Thakkar; Michael Hindle; Umesh R. Desai (45-51).
► Sulfated LMWL fractions were isolated by dynamic thrombin affinity chromatography. ► The fractions differ significantly in their biophysical and biochemical properties. ► One fraction of the three studied exhibited potent antagonism of clotting. ► This fraction selectively attenuated the function of extrinsic pathway. ► Identified the 1st LMWL structure that exhibits antagonism of the extrinsic pathway.Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4m/z, which most probably arise from variably functionalized β-O4―β-β-linked trimers and/or a β-O4―β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants.
Keywords: Sulfated lignins; Heparin; Heparan sulfate; Anticoagulation; Enzyme inhibition; Dynamic affinity chromatography; Mass spectrometry;

► Simultaneously determine morinidazole and its four metabolites in human plasma. ► Gradient elution was used to obtain the resolution of two N+-glucuronide isomers. ► The potential interference from the in-source dissociation of the conjugates was avoided. ► The method was applied to clinical pharmacokinetic studies of morinidazole injection.Morinidazole is a new third-generation 5-nitroimidazole antimicrobial drug. To investigate the pharmacokinetic profiles of morinidazole and its major metabolites in humans, a liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous determination of morinidazole, its N-oxide metabolite (M4-1), a sulfate conjugate (M7), and two diastereoisomeric N +-glucuronides (M8-1 and M8-2) in human plasma. A simple acetonitrile-induced protein precipitation was employed to extract five analytes and internal standard metronidazole from 50 μL human plasma. To avoid the interference from the in-source dissociation of the sulfate and achieve the baseline-separation of diastereoisomeric N +-glucuronides, all the analytes were separated from each other with the mobile phase consisting of 10 mM ammonium formate and acetonitrile using gradient elution on a Hydro-RP C18 column (50 mm × 2 mm, 4 μm) with a total run time of 5 min. The API 4000 triple quadrupole mass spectrometer was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. The developed method was linear in the concentration ranges of 10.0–12,000 ng/mL for morinidazole, 1.00–200 ng/mL for M4-1, 2.50–500 ng/mL for M7, 3.00–600 ng/mL for M8-1, and 10.0–3000 ng/mL for M8-2. The intra- and inter-day precisions for each analyte met the accepted value. Results of the stability of morinidazole and its metabolites in human plasma were also presented. The method was successfully applied to the clinical pharmacokinetic studies of morinidazole injection in healthy subjects, patients with moderate hepatic insufficiency, and patients with severe renal insufficiency, respectively.
Keywords: Liquid chromatography–tandem mass spectrometry; Morinidazole; Conjugated metabolites; N-Oxide metabolite; Pharmacokinetics; Plasma;

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics by Joseph M. Boutilier; Hunter Warden; Alan A. Doucette; Peter D. Wentzell (59-66).
► The isotope effect (D0 vs D4) in dimethyl labelling of peptide pairs is examined. ► Four replicates of 71 peptide pairs are used to assess retention time differences. ► Retention differences are statistically significant, but small and inconsequential. ► Computational results are consistent with observations for other labelling methods. ► Dimethyl labelling is a viable alternative for relative peptide quantitation.The differential separation of deuterated and non-deuterated forms of isotopically substituted compounds in chromatography is a well-known but not well-understood phenomenon. This separation is relevant in comparative proteomics, where stable isotopes are used for differential labelling and the effect of isotope resolution on quantitation has been used to disqualify some deuterium labelling methods in favour of heavier isotopes. In this work, a detailed evaluation of the extent of isotopic separation and its impact on quantitation was performed for peptides labelled through dimethylation with H2/D2 formaldehyde. The chromatographic behaviour of 71 labelled peptide pairs from quadruplicate tryptic digests of bovine serum albumin were analysed, focusing on differences in median retention times, resolution, and relative quantitation for each peptide. For 94% of peptides, the retention time difference (heavy-light) was less than 12 s with a median value 3.4 s. With the exception of a single anomalous pair, isotope resolution was below 0.6 with a median value 0.11. Quantitative assessment indicates that the bias in ratio calculation introduced by retention time shifts is only about 3%, substantially smaller than the variation in ratio measurements themselves. Computational studies on the dipole moments of deuterated labels indicate that these results are consistent with literature suggestions that retention time shifts are inversely related to the polarity of the label. This study suggests that the incorporation of deuterium isotopes through peptide dimethylation at amine residues is a viable route to proteome quantitation.
Keywords: Dimethyl labelling; Isotopic effects; Deuterium isotope effect; Comparative proteomics; Relative quantitation; Formaldehyde;

► HFLM-SPME was proposed for the determination of NSAIDs in urine samples. ► PEG-g-MWCNTs was used as extraction phase to prepare the titania sol–gel SPME fiber. ► The HFLM-SPME method, integrating the advantages of SPME based sol–gel and HF-LPME. ► This method has a considerably low LOD and a relatively wide linear rang.A new rapid, simple and effective cleanup procedure is demonstrated for the determination of ibuprofen, naproxen and diclofenac in urine samples by using hollow-fiber liquid membrane-protected solid-phase microextraction (HFLM-SPME) based on sol–gel technique and gas chromatography–flame ionization detector (GC–FID). In this technique, a sol–gel coated fiber was protected with a length of porous polypropylene hollow fiber membrane which was filled with water-immiscible organic phase. Subsequently the whole device was immersed into urine sample for extraction. Poly(ethylene glycol) (PEG) grafted onto multi-walled carbon nanotubes (PEG-g-MWCNTs) was used as extraction phase to prepare the sol–gel SPME fiber. Important parameters influencing the extraction efficiency such as desorption temperature and time, organic solvent, extraction temperature and time, pH, stirring speed and salt effect were investigated and optimized. Under the optimal conditions, the method detection limits (S/N = 3) were in the range of 0.03–0.07 ng mL−1 and the limits of quantification (S/N = 10) between 0.08 and 0.15 ng mL−1. Relative standard deviations for intra-day and inter-day precisions were 4.8–9.0% and 4.9–8.1%, respectively. Subsequently, the method was successfully applied to human urine fractions after administration of ibuprofen, naproxen and diclofenac.
Keywords: Hollow-fiber liquid membrane-protected solid-phase microextraction; Multi-walled carbon nanotubes; Sol–gel technology; Non-steroidal anti-inflammatory drugs; Urine samples;

► An LC–MS/MS method was validated in ocular matrix. ► Sodium adducts transition was utilized for quantification of rapamycin, and was stable for 4 weeks in the matrix. ► This method was successfully applied to estimate the rapamycin in the rabbit eye. ► This system demonstrates that 0.2% rapamycin nanomicellar formulation can successfully delivers therapeutic drug levels in the eye.A novel, fast and sensitive 3200 QTRAP LC–MS/MS method was validated for rapamycin analysis in the rabbit eye following 0.2% administration of nanomicellar eye drop formulation. The LC–MS/MS technique was developed with electrospray ionization (ESI) in positive mode. Rapamycin was extracted from individual eye tissues and fluids by a simple protein precipitation method. Samples were reconstituted in 200 μL of 80% of acetonitrile in water containing 0.05% formic acid. Twenty microliter of the sample was injected on LC–MS/MS. Chromatographic separations was achieved on reversed phase C 8 Xterra column, 50 mm × 4.6 mm, 5 μm. Multiple reactions monitoring (MRM) transition m/z 936.6/409.3 for rapamycin and 734.4/576.5 for erythromycin were employed as internal standard. The calibration curves were linear r 2  > 0.9998 over the concentration range from 2.3 ng/mL to 1000.0 ng/mL. Rapamycin was found to be stable in ocular tissue homogenates for 6 weeks at a refrigerated −80 °C and −20 °C temperatures. Rapamycin concentration was found to be 2260.7 ± 507.1 (mean ± S.D.) ng/g tissue and 585.5 ± 80.1 (mean ± S.D.) ng/g tissue in the cornea and iris ciliary muscle, respectively. This method has two advantages. First, a volatile base was used in the extraction procedure, which is easy to evaporate and generate consistent results. Second, the sodium adduct is employed that was stable in non-ammoniated mobile phase. The method demonstrates that absorption of rapamycin by a topical application of 0.2% rapamycin nanomicellar formulation generates therapeutically effective concentrations in the anterior segment of the eye.
Keywords: Rapamycin (Sirolimus); 3200 QTRAP LC–MS/MS bioanalytical method validation; Protein precipitation extraction; Rabbit anterior ocular tissue distribution study; 0.2% rapamycin nanomicellar formulation;

► Piperphentonamine hydrochloride for injection is an original new drug firstly used in human, which belongs to calcium sensitizers. ► A LC/MS/MS method to determine the concentrations of piperphentonamine and metabolites M1 and M6 in human plasma and urine was established in this paper. ► This method was adopted to perform quantitative analysis of piperphentonamine and metabolites in plasma and urine of healthy Chinese subjects. ► The pharmacokinetic characteristics of single intravenous bolus of piperphentonamine and metabolites in three dosage groups in healthy Chinese subjects were evaluated.Piperphentonamine hydrochloride (PPTA) is a new calcium sensitizer. A liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for determination of piperphentonamine and its metabolites M1 and M6 was developed for the first time and applied to a pharmacokinetics study. Protein precipitation was used for pre-treatment of plasma samples, and solid phase extraction method was used for pre-treatment of urine samples. The chromatographic separation was achieved on a C18 column using gradient elution in this study: A: 1% acetic acid aqueous solution, and B: acetonitrile. The whole analysis lasted for 10.5 min and the gradient flow rate was 0.25 mL/min constantly. The detection was performed of a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via a positive electrospray ionization source. The results were that the m/z ratios of monitored precursor ions and product ions of PPTA, M1 and M6 were 354.0 → 191.8, 356.0 → 148.7 and 358.0 → 148.7, respectively. From the standard curve, the concentration ranges of both PPTA and M1 in blood and urine samples were 0.1–500 ng/mL and 0.1–200 ng/mL, respectively; the concentration ranges of M6 in blood sample and urine sample were 0.2–500 ng/mL and 0.2–200 ng/mL, respectively; and the correlation coefficient of standard curve was r  > 0.99. A total of 31 healthy Chinese subjects participated in the pharmacokinetic study of single bolus intravenous injection of piperphentonamine hydrochloride. They were divided into three dosage groups and given 0.2, 0.4 and 0.6 mg/kg of PPTA. After drug administration, concentrations of PPTA, M1 and M6 in human plasma and urine samples were determined to evaluation the pharmacokinetic characteristics of PPTA and its metabolites M1 and M6.
Keywords: Piperphentonamine hydrochloride; LC/MS/MS; Metabolite; Pharmacokinetics; Phase I clinical study;

Capillary electrophoresis to quantitate gossypol enantiomers in cotton flower petals and seed by Sergey Vshivkov; Egor Pshenichnov; Zamira Golubenko; Alik Akhunov; Shadman Namazov; Robert D. Stipanovic (94-97).
► Gossypol occurs as a mixture of enantiomers in cottonseed. ► (−)-Gossypol is more toxic to non-ruminant animals. ► Plant breeders are developing plants with a low percent of (−)gossypol. ► A capillary electrophoresis method was developed to quantitate the enantiomers. ► Breeders can use this method to select plants with the low (−)gossypol seed trait.Gossypol is a toxic compound that occurs as a mixture of enantiomers in cotton plant tissues including seed and flower petals. The (−)-enantiomer is more toxic to non-ruminant animals. Efforts to breed cottonseed with a low percentage of (−)-gossypol requires determination of the (+)- to (−)-gossypol ratio in seed and flower petals. We report a method to quantitatively determine the total gossypol and percent of its enantiomers in cotton tissues using high performance capillary electrophoresis (HPCE). The method utilizes a borate buffer at pH 9.3 using a capillary with internal diameter of 50 μm, effective length of 24.5 cm, 15 kV and cassette temperature of 15 °C. This method provides high accuracy and reproducible results with a limit of detection of the individual enantiomers of less than 36 ng/mL providing base line separation in less than 6 min.
Keywords: (+)-Gossypol; (−)-Gossypol; Cottonseed; Capillary electrophoresis;

► A microfractionation bioactivity-based fingerprint of A. scholaris was established. ► This method was coupled with two dual-luciferase reporter assay systems. ► NF-κB inhibitors and β2AR agonists can be screened simultaneously in one process. ► Three dual-functional indole alkaloids with dual-target were found. ► The strategy is suitable for identifying dual-target compounds in complex systems.Traditional Chinese medicines (TCMs) are generally considered complementary or alternative remedies in most Western countries. The constituents of TCMs are hard to define, and their efficacy is difficult to appraise. Thus, the development of suitable methods for evaluating the relationship between bioactivity and the chemical makeup of complex TCM mixtures remains a great challenge. In the present work, the bioactivity-integrated fingerprints of alkaloidal leaf extracts of Alstonia scholaris, a folk medicinal herb for chronic respiratory diseases, were established by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF). This method was coupled with two dual-luciferase reporter assay systems to show nuclear factor-κB (NF-κB) inhibition and β2 adrenergic receptor (β2AR) activation. Using UPLC-Q/TOF, 18 potential candidates were identified according to unique mass spectrometric fragmentation. After in vitro biological evaluation, several indole alkaloids with anti-inflammatory and anti-asthmatic properties were found, including akuammidine, (E)-alstoscholarine, and (Z)-alstoscholarine. Compared with conventional fingerprints, the microfractionation based bioactivity-integrated fingerprints that contain both chemical and bioactivity details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that dual bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.
Keywords: Dual-bioactivity-integrated fingerprinting; Constituent identification; Nuclear factor-κB inhibitor; β2 Adrenergic receptor agonist; Ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry;

Insulin related compounds and identification by Cheol-Ki Min; Jin-Won Lee; Chang-Kyu Kim; Seong Hwan Kim; Sang Yeol Lee; Sang-Myung Lee; Young-Jin Son (105-112).
► We report six human insulin-related compounds from two different expression strains. ► Insulin-related compounds were purified and identified using analytical methods. ► An analytical HPLC method was introduced to detect desthreonine human insulin, which is a major impurity during human insulin production. ► The formation mechanisms of insulin-related compounds were studied based on the identities of the related compounds. ► Taken together, our results present several insulin-related compounds and effective analytical methods for detecting them.Insulin-related compounds (IRCs), which originate during the expression and purification of human insulin using recombinant Escherichia coli, were purified and identified. We investigated the identity of IRCs and their origin. We also presented methods for inhibiting IRC formation. The strains used in this report were E. coli B5K and E. coli H27R. E. coli B5K had a 6-amino acid-fused peptide at the N-terminus of proinsulin, and E. coli H27R had a 28-amino acid-fused peptide at the N-terminus of proinsulin. We investigated the identity of IRCs and their origin by mainly using High Performance Liquid Chromatography (HPLC). The well-known IRCs, desamido human insulin and desthreonine human insulin, formed in both strains. In addition to these two IRCs, the B5K strain produced three different IRCs, ArgA(0)-insulin (IRC 1), prepeptide-insulin (IRC 2), and GluA(22)-insulin (IRC 3). The amounts of IRC 1, IRC 2, IRC 3 were approximately 0.1–0.3% after final purification step. Among these IRCs, ArgA(0)-insulin, prepeptide-insulin, and desthreonine insulin originated from incomplete enzyme reaction. GluA(22)-insulin was formed when we used a double stop codon during the expression of preproinsulin; that is, it was formed by the misreading of the first stop codon through the amber mutation. The major IRCs of the H27R strain were human insulin fragment (B1–B21) (IRC 4), and A9(Ser → Asn) amino acid single mutation human insulin (IRC 5), ArgB(31)-insulin (IRC 6). Human insulin fragment (B1–B21) was formed by β-mercaptoethanol, which was added during refolding. It formed when the disulfide bonds between A-chain and B-chain of human insulin were cut by β-mercaptoethanol, followed by cleavage of the B-chain by trypsin and carboxypeptidase B. A9(Ser → Asn) amino acid single mutation human insulin originated from the mistranslation of A9 serine, such that asparagine was translated instead of serine. ArgB(31)-insulin originated from incomplete enzyme reaction. The amount of IRC 4 was 10–15% after enzyme reaction. The amounts of IRC 5, IRC 6 were around 0.2% after final purification step. We present methods for inhibiting the formation of IRCs by controlling the amount of enzyme, controlling the rate of enzyme reaction, using a single stop codon, using hydrogen peroxide (H2O2) to inhibit β-mercaptoethanol, and modifying the A9 codon.
Keywords: Insulin related compounds; Identification; Chromatography;

► Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for PVC. ► DEHP metabolites have been proposed as markers of the misuse of blood transfusion. ► A method to quantify five DEHP metabolites has been developed and validated. ► Cutoff concentrations were proposed to determine suspicions of a possible transfusion. ► The method is proposed as screening test for transfusion detection in doping control.Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for polyvinyl chloride, which is found in a large variety of products, including most of the bags used for blood storage because of its protective role on erythrocytes survival. DEHP metabolites have been recently proposed as markers of the misuse of blood transfusion in athletes. In this study, a method to quantify the main five DEHP metabolites in urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an enzymatic hydrolysis with β-glucuronidase from Escherichia coli followed by an acidic extraction with ethyl acetate. The hydrolysed extracts were analysed by ultraperformance liquid chromatography tandem mass spectrometry. Isotope labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of all the metabolites was achieved in a total run time of 10 min, using a C18 column and a mobile phase containing deionized water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min−1. Detection of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive and negative ion modes. The method was validated for quantitative purposes. Extraction recoveries were greater than 90% and the limits of quantitation ranged from 1.2 to 2.6 ng mL−1. Intra-day precisions were better than 8% for all metabolites while inter-assay precisions were better than 12%. Concentrations of DEHP metabolites were measured in a control group (n  = 30, subjects reflecting the common environmental DEHP exposure), and in sportsmen (n  = 464), to evaluate population distribution exposure to DEHP. Additionally, threshold concentrations indicating outliers of common exposure for DEHP metabolites are proposed.
Keywords: Blood doping; Transfusion; Di-(2-ethylhexyl)phthalate (DEHP); Plasticizers; Ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS); Doping control;

The principle of pooled calibrations and outlier retainment elucidates optimum performance of ion chromatography by Jens E.T. Andersen; Maria Mikolajczak; Katarzyna Olga Wojtachnio-Zawada; Henrik Vigan Nicolajsen (122-127).
► Outliers of IC should be retained in order to secure correct level of uncertainty. ► Pooled calibrations provide excellent correspondence between uncertainties. ► A new method for the determination of the full-calibration range in IC is proposed. ► The contents determined by IC do not correlate with the sensitivity. ► RSDs of unknowns correspond well to the uncertainty predicted by Horwitz.A principle with quality assurance of ion chromatography (IC) is presented. Since the majority of scientists and costumers are interested in the determination of the true amount of analyte in real samples, the focus of attention should be directed towards the concept of accuracy rather than focussing on precision. By exploiting the principle of pooled calibrations and retainment of all outliers it was possible to obtain full correspondence between calibration uncertainty and repetition uncertainty, which for the first time evidences statistical control in experiments with ion chromatography. Anions of bromide were analysed and the results were subjected to quality assurance (QA). It was found that the limit of quantification (LOQ) was significantly underestimated by up to a factor of 30 with respect to the determination of concentration of unknowns. The concept of lower-limit of analysis (LLA) and upper-limit of analysis (ULA) were found to provide more acceptable limits with respect to reliable analysis with a limited number of repetitions. An excellent correspondence was found between calibration uncertainty and repetition uncertainty. These findings comply with earlier investigations of method validations where it was found that the principle of pooled calibrations provides a more realistic picture of the analytical performance with the drawback, however, that generally higher levels of uncertainties should be accepted, as compared to contemporary literature values. The implications to the science analytical chemistry in general and to method validations in particular are discussed.
Keywords: Ion chromatography; Quality assurance; Internal standard; Certified reference material;

Analytical method for the sensitive determination of major di-(2-propylheptyl)-phthalate metabolites in human urine by Wolfgang Gries; Dietmar Ellrich; Katja Küpper; Birgit Ladermann; Gabriele Leng (128-136).
► DPHP-metabolites OH-MPHP, oxo-MPHP and cx-MPHxP are measured in human urine. ► GC–HRMS, GC–MS/MS, HPLC–MS/MS methods are presented. ► Detection limits range between 0.05 and 0.2 μg/L urine for each metabolite. ► Sensitive determination of DPHP metabolites in environmental medicine is possible.Di-(2-propylheptyl)-phthalate (DPHP) is a specific phthalic acid ester of isomeric C10 alcohols. It is classified as high molecular weight phthalate and marketed as plasticizer for polyvinyl chloride (PVC). The increase of its production volume and its wide field of application suggest a possible background exposure of the human population as found for other phthalates, making suitable analytical methods necessary. The aim of the presented analytical report is the sensitive and selective determination of the three major DPHP metabolites mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP), mono-2-(propyl-6-oxoheptyl)-phthalate (oxo-MPHP) and mono-2-(propyl-6-carboxy-hexyl)-phthalate (cx-MPHxP) in human urine. Most of the published analytical methods for phthalate metabolites use high pressure liquid chromatography tandem mass spectrometry (HPLC–MS/MS). The methods presented here allow a comparison of chromatographic separation between HPLC–MS/MS and gas chromatography high resolution mass spectrometry (GC–HRMS), which is useful to distinguish between DPHP and DIDP. The enhanced detection limits range between 0.05–0.1 μg/L for GC–HRMS and 0.1–0.2 μg/L for HPLC–MS/MS.
Keywords: Biomonitoring; DPHP metabolites; Phthalates; Urine; GC–MS; LC–MS/MS;

► A simple and rapid MI-MSPD method for determination of melamine from milk. ► Water-compatible MIP was obtained by cyromazine as dummy template in methanol–water solution. ► Rapid screening of melamine in milk, while interferences from milk matrix were eliminated. ► Extraction efficiency was markedly increased with reduced equilibrium time. ► This method improved the selectivity and eliminated the effect of template leakage on quantitative analysis.A simple, convenient and high selective molecularly imprinted matrix solid-phase dispersion (MI-MSPD) using water-compatible cyromazine-imprinted polymer as adsorbent was proposed for the rapid screening of melamine from bovine milk coupled with liquid chromatography-ultraviolet detection. The molecularly imprinted polymers (MIPs) synthesized by cyromazine as dummy template and reformative methanol–water system as reaction medium showed higher affinity and selectivity to melamine, and so they were applied as the specific dispersant of MSPD to extraction of melamine and simultaneously eliminate the effect of template leakage on quantitative analysis. Under the optimized conditions, good linearity was obtained in a range of 0.24–60.0 μg g−1 with the correlation coefficient of 0.9994. The recoveries of melamine at three spiked levels were ranged from 86.0 to 96.2% with the relative standard deviation (RSD) ≤ 4.0%. This proposed MI-MSPD method combined the advantages of MSPD and MIPs, and could be used as an alternative tool for analyzing the residues of melamine in complex milk samples.
Keywords: Matrix solid-phase dispersion; Dummy imprinted polymers; Melamine; High performance liquid chromatography; Milk samples;

► Simultaneous separation and purification of baicalin and wogonoside by macroporous resin. ► Process parameters were optimized for the separation procedure. ► HPD-100 resin was successfully applied to obtain products of baicalin and wogonoside with high contents. ► The developed method was validated by a laboratory preparative-scale separation.In this study, resin adsorption as a means to separate and purify baicalin and wogonoside from extracts of Scutellaria baicalensis was investigated. Among the ten tested resins, the non-polar resin HPD-100 offered the best adsorption and desorption properties. Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures, and the equilibrium experimental data were well fitted to the two isotherms. Column packed with HPD-100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. After one round treatment with HPD-100 resin, the contents of baicalin and wogonoside were 3.6-fold and 12.0-fold increased with recovery yields of 85.7% and 65.6%, respectively. In addition, a laboratory preparative-scale separation was carried out under the final conditions. The results showed that the preparative separation of baicalin and wogonoside can be easily and efficiently achieved via adsorption and desorption on HPD-100 resin. The developed method is a promising basis for large-scale preparation of baicalin and wogonoside from S. baicalensis extracts.
Keywords: Baicalin; Wogonoside; Macroporous resins; Purification; Adsorption chromatography;

Preparative separation of glycoalkaloids α-solanine and α-chaconine by centrifugal partition chromatography by Jacques Attoumbré; David Lesur; Philippe Giordanengo; Sylvie Baltora-Rosset (150-154).
► α-Solanine and α-chaconine were extracted from Solanum tuberosum cv. Pompadour. ► CPC was used to develop an efficient preparative separation procedure. ► The efficiency of the separation depended on the alkaloid. ► The highest purity and yield were achieved for α-chaconine.The main glycoalkaloids of a commercial potato cultivar, α-chaconine and α-solanine, were extracted from sprouts of Solanum tuberosum cv. Pompadour by a mixture of MeOH/H2O/CH3COOH (400/100/50, v/v/v). In these conditions, 2.8 ± 0.62 g of crude extract were obtained from 50 g of fresh sprouts and the total glycoalkaloid content was determined by analytical HPLC at 216.5 mg/100 g. α-Chaconine and α-solanine were separated in a preparative scale using centrifugal partition chromatography (CPC). In a solvent system composed of a mixture of ethyl acetate/butanol/water (15/35/50, v/v/v), α-chaconine (54 mg) and α-solanine (15 mg) were successfully isolated from the crude extract in one step of purification. The purity of isolated compounds was determined to be higher than 92% by HPLC analysis.
Keywords: Glycoalkaloids; α-Chaconine; α-Solanine; Centrifugal partition chromatography (CPC); High performance liquid chromatography (HPLC);

A rapid quantitative method of carisoprodol and meprobamate by liquid chromatography–tandem mass spectrometry by Shannon Essler; Kerry Bruns; Michael Frontz; J. Rod McCutcheon (155-160).
► Carisoprodol and meprobamate are commonly encountered drugs in forensic toxicology. ► In this method, sample preparation can be performed with a minimum of materials and effort. ► Using LC/MS/MS provides less need for a clean extract and adds speed and specificity to the analysis. ► The method uses deuterium labeled internal standards for both analytes.The identification and quantitation of carisoprodol (Soma) and its chief metabolite meprobamate, which is also a clinically prescribed drug, remains a challenge for forensic toxicology laboratories. Carisoprodol and meprobamate are notable for their widespread use as muscle relaxants and their frequent identification in the blood of impaired drivers. Routine screening is possible in both an acidic/neutral pH screen and a traditional basic screen. An improvement in directed testing quantitations was desirable over the current options of an underivatized acidic/neutral extraction or a basic screen, neither of which used ideal internal standards. A new method was developed that utilized a simple protein precipitation, deuterated internal standards and a short 2-min isocratic liquid chromatography separation, followed by multiple reaction monitoring with tandem mass spectrometry. The linear quantitative range for carisoprodol was determined to be 1–35 mg/L and for meprobamate was 0.5–50 mg/L. The method was validated for specificity and selectivity, matrix effects, and accuracy and precision.
Keywords: Postmortem toxicology; Human performance toxicology; Liquid chromatography–tandem mass spectrometry; Carisoprodol; Meprobamate; Method validation;