Journal of Chromatography B (v.902, #C)

► Silymarin extract contains numerous bioactive flavonolignans and flavonoids. ► Analysis of silychristin, silydianin, silybin A and B, isosilybin A and B, and taxifolin. ► Pure standard compounds were utilized for absolute quantification. ► The lower limit of quantification was 2 ng/ml for each analyte. ► Method was applied to human plasma samples from subjects treated with Legalon®.Silymarin, an extract of crushed achenes of the milk thistle plant Silybum marianum is a multi-constituent mixture, 70–80% of which consists of a complex assortment containing the flavonolignans silybin A and B, isosilybin A and B, silydianin, and silychristin, and the flavonoid taxifolin. To date, numerous pharmacological actions of the silymarin extract have been documented in the biomedical literature, including hepatoprotective, anti-inflammatory, anti-tumor, and anti-fibrotic activities. The present study describes a novel liquid chromatographic–tandem mass spectrometric method for simultaneous analysis of silychristin, silydianin, silybin A and silybin B, isosilybin A and isosilybin B, and taxifolin in human plasma employing liquid–liquid extraction. This assay provides excellent resolution of the individual silymarin constituents via utilization of a 100 A 250 mm × 2 mm, 5 μm C18 column with the mobile phase consisting of 51% methanol, 0.1% formic acid, and 10 mM ammonium acetate. The lower limit of quantification was 2 ng/ml for each constituent. Calibration curves were linear over the range from 2 ng/ml to 100 ng/ml for all analytes (r 2  > 0.99). The intra- and inter-day accuracies were 91–106.5% and 95.1–111.9%, respectively. The intra- and inter-day precision was within 10.5%. Additionally, recovery, stability, and matrix effects were fully validated as well. This method was successfully applied to human plasma samples from subjects treated with the milk thistle extract Legalon®.
Keywords: Milk thistle; Silymarin; Silybin; Taxifolin; LC–MS/MS;

► A novel LC/MS/MS-based targeted proteomics assay was developed and validated. ► Protein digestion efficiency and surrogate peptide selection were discussed. ► The LC/MS/MS assay was compared with a commercial immunoturbidimetric method. ► Clinical monitoring of prospective protein biomarkers could be achieved by LC/MS/MS.Currently, the absolute quantification of human transferrin (hTRF) is based on several techniques other than mass spectrometry. Although these techniques provide valuable information on protein levels and can be extremely sensitive, they often lack the specificity and reproducibility that can be provided by mass spectrometry. In this study, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) based targeted proteomics assay was developed and validated for the determination of transferrin in human serum. We selected the tryptic peptide 108EDPQTFYYAVAVVK121 as the surrogate analyte for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Sample cleanup and enrichment were achieved using solid phase extraction. The validated calibration range was from 500 to 5000 ng/mL. The intra- and inter-day precisions were less than 4.9% and 9.0%, respectively. The bias for the quality control (QC) samples was less than 5.4%. Finally, this assay was successfully applied to the quantitative analysis of transferrin in clinical samples. The obtained values were assessed by independently measuring transferrin in the same samples using a commercially available immunoturbidimetric assay. As a result, the absolute concentrations determined by the LC/MS/MS assay compared well with those obtained with the immunoturbidimetric method; however, the LC/MS/MS assay afforded more reliable transferrin values at low concentrations.
Keywords: Human transferrin; Liquid chromatography–tandem mass spectrometry; Targeted proteomics; Stable isotopic labeling; Method validation; Clinical application;

Development, validation and application of a sensitive LC–MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium by Sandra Roche; Louise Sewell; Justine Meiller; Kasper Pedersen; Rajesh Rajpal; Peter O’Gorman; Martin Clynes; Robert O’Connor (16-26).
► We developed a sensitive LCMS method for thalidomide with an LOQ of 3 ng/mL in serum. ► The sample preparation, LLE, gives higher sample recovery than previous methods. ► In patient serum we detected thalidomide levels ranging from 25 ng/mL to 1407 ng/mL. ► We quantified thalidomide degradation in vitro with 50% remaining after 2 h.A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100 μL), cells (2.5 × 105), or cell culture media (100 μL) by LLE and separated on a Prodigy C18 (150 mm × 4.0 mm, 5 μm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5 mL/min, with umbelliferone (600 ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05–20 μg/mL) and in vitro cells (0.78–50 ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3 ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2 h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.
Keywords: Thalidomide; LC–MS/MS; LLE; Multiple myeloma; Cell culture; Stability; Thalidomide pharmacokinetics; Compliance;

► Indolinone is stable in rat plasma stored below −65 °C during 190 days. ► Method in rat plasma for indolinone is specific, selective, precise and accurate. ► Indolinone showed a short half-life and a relatively high clearance in rat plasma.The natural product (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified some years ago as a nanomolar inhibitor of FcɛRI-receptor dependent mast cell degranulation. To further explore the potential of the compound, we established an UPLC-MS/MS assay for dosage in rat plasma. The method was fully validated according to FDA Guidance for industry. Results of this validation and long term stability study demonstrate that the method in lithium heparinized rat plasma is specific, accurate, precise and capable of producing reliable results according to recommendations of international guidelines. The method was validated with a LLOQ of 30.0 ng/mL and an ULOQ of 3000 ng/mL. The response versus concentration data were fitted with a first order polynomial with 1/X 2 weighting. No matrix effect was observed when using three independent sources of rat plasma. The average extraction recovery was consistent over the investigated range. This validation in rat plasma demonstrated that indolinone was stable for 190 days when stored below −65 °C; for 4 days at 10 °C in the autosampler; for 4 h at RT, and during three successive freeze/thaw cycles at −65 °C. Preliminary pharmacokinetic data were obtained in male Sprague–Dawley rats (2 mg/kg BW i.v.). Blood samples taken from 0 to 12 h after injection were collected, and data analyzed with WinNonlin. A short half-life (4.30 ± 0.14 min) and a relatively high clearance (3.83 ± 1.46 L/h/kg) were found.
Keywords: Indolinone; Anti-allergic; Isatis tinctoria L.; UPLC-MS/MS; Validation; Pharmacokinetic study;

A fully integrated multi-column system for abundant protein depletion from serum/plasma by Dariusz J. Janecki; Steven C Pomerantz; Eric J. Beil; Jennifer F. Nemeth (35-41).
► A conventional HPLC system set-up for automated serum/plasma depletion. ► The system uses two immunoaffinity columns in series (Seppro IgY14 and SuperMix). ► The autosampler is dual-purposed for both injection and fraction collection. ► The system significantly speeds up the sample preparation for proteomic workflow.This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125 psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200–300 proteins (Seppro SuperMix). The autosampler was dual-purposed for both injection and fraction collection. Both the flow-through and SuperMix bound proteins were collected in an automated fashion. Three samples could be depleted consecutively before the system required user intervention, and up to nine samples could be depleted within a 24 h period. This study documents the validation of the instrument performance with a 90-patient sample set, demonstrating overall CVs for 86 of the 90 samples to be within the 95% confidence intervals. Additionally, there was excellent reproducibility within the same patient (biological replicates) across days.
Keywords: Serum depletion; Automation; Fraction collection; Immunoaffinity columns;

Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening by Sevasti Karampela; Ioanna Vardakou; Ioannis Papoutsis; Artemis Dona; Chara Spiliopoulou; Sotirios Athanaselis; Constantinos Pistos (42-46).
► We develop simpler and faster sample preparation method for the determination of benzodiazepines in urine. ► We overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods. ► We overcome false negative/false positive results when using immunoassay methods for benzodiazepines. ► The method is applicable in real clinical and forensic samples.A simple and rapid LC/MS method with direct injection analysis was developed and validated for the identification and quantification of ten benzodiazepines (flunitrazepam, nordiazepam, diazepam, 7-aminoflunitrazepam, flurazepam, bromazepam, midazolam, alprazolam, temazepam and oxazepam) in human urine using diazepam-d5 as internal standard (IS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as diluted urine samples were directly injected into LC/MS system. Electrospray ionization in positive mode using selected ion monitoring was chosen for the identification and quantification of the analytes. The linear range was 50–1000 ng/mL for each analyte, with square correlation coefficient (r 2) ≥ 0.981. Interday and intraday errors were found to be ≤5.72%. The LC/MS method was applied at ten real samples found initially to be positive and negative, using immunoassay technique. Finally the results were confirmed with GC/MS. The method demonstrates simplicity and fast sample preparation, accuracy and specificity of the analytes which make it suitable for replacement of immunoassay screening in urine avoiding thus false negative/false positive results. Using this method, laboratories may overcome the problem of high cost instrumentation such as LC–MS/MS by providing similar sensitivity and specificity with other methods.
Keywords: Benzodiazepines; Human urine; LC/MS; Direct injection; Screening;

Liquid chromatography–mass spectrometric determination of losartan and its active metabolite on dried blood spots by R. Nageswara Rao; S. Satyanarayana Raju; R. Mastan Vali; G. Girija Sankar (47-54).
► A simple isocratic LC–MS/MS method was developed and validated for losartan and its active metabolite. ► First dried blood spot report on losartan and it active metabolite as sample collection technique. ► Stability of analytes on FTA cards was also studied. ► Good LC resolution was obtained between the analytes and IS.A simple and rapid quantitative bioanalytical liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for simultaneous determination of losartan and its active metabolite, losartan carboxylic acid on rat dried blood spots was developed and validated as per regulatory guidelines. Losartan and its metabolite were extracted from dried blood spots using 50% aqueous methanol and separated on Waters XTerra® RP18 (250 mm × 4.6 mm, 5 μm) column using mobile phase composed of 40% acetonitrile and 60% aqueous ammonium acetate (10 mM). The eluents were monitored using ESI tandem mass spectrometric detection with negative polarity in MRM mode using ion transitions m/z 421.2 → 179.0, m/z 435.3 → 157.0 and m/z 427.3 → 193.0 for losartan, losartan carboxylic acid and Irbesartan (internal standard), respectively. The method was validated over the linear range of 1–200 ng/mL and 5–1000 ng/mL with lower limits of quantification of 1.0 ng/mL and 5.0 ng/mL for losartan and losartan carboxylic acid, respectively. Inter and intra-day precision and accuracy (Bias) were below 5.96% and between −2.8 and 1.5%, respectively. The mean recoveries of the analytes from dried blood spots were between 89% and 97%. No significant carry over and matrix effects were observed. The stability of stock solution, whole blood, dried blood spot and processed samples were tested under different conditions and the results were found to be well within the acceptable limits. Additional validation parameters such as influence of hematocrit and spot volume were also evaluated and found to be well within the acceptable limits.
Keywords: Losartan (LOS); Losartan carboxylic acid (LCA); Dried blood spots (DBS); LC–MS/MS; Stability; Validation;

Characterization of taste-active compounds of various cherry wines and their correlation with sensory attributes by Yunwei Niu; Xiaoming Zhang; Zuobing Xiao; Shiqing Song; Chengsheng Jia; Haiyan Yu; Lingling Fang; Chunhua Xu (55-60).
► Four sensory attributes of 5 cherry wines were evaluated. ► Thirty-three compounds were correlated to sensory attributes and wine samples through PLSR. ► Seventeen compounds mostly contributed to sensory attributes of cherry wines.Five cherry wines exhibiting marked differences in taste and mouthfeel were selected for the study. The taste and mouthfeel of cherry wines were described by four sensory terms as sour, sweet, bitter and astringent. Eight organic acids, seventeen amino acids, three sugars and tannic acid were determined by high performance liquid chromatography (HPLC). Five phenolic acids were determined by ultra performance liquid chromatography coupled with mass spectrometry (UPLC–MS). The relationship between these taste-active compounds, wine samples and sensory attributes was modeled by partial least squares regression (PLSR). The regression analysis indicated tartaric acid, methionine, proline, sucrose, glucose, fructose, asparagines, serine, glycine, threonine, phenylalanine, leucine, gallic acid, chlorogenic acid, vanillic acid, arginine and tannic acid made a great contribution to the characteristic taste or mouthfeel of cherry wines.
Keywords: Cherry wine; Taste-active compounds; Sensory attribute; High performance liquid chromatography; Ultra performance liquid chromatography coupled with mass spectrometry; Partial least squares regression analysis;

Pharmacokinetics of conjugated metabolites in rat plasma after oral administration of tectoridin by Jialin Qu; Jie Gao; Jiahong Sun; Lin Zhang; Toshiaki Makino; Dan Yuan (61-69).
► A new glucuronide-sulfate diconjugate was isolated and structurally determined for the first time. ► Three conjugate metabolites were simultaneously identified and quantified in rat plasma by HPLC–DAD and LC/TOF/MS. ► It provides helpful information for the potential therapeutic application of tectoridin-containing phytopharmacueticals.Tectoridin is a major isoflavone found in the flowers of Pueraria thomsonii Benth. It possesses estrogenic, hypoglycemic, anti-oxidant, and anti-inflammatory activities. In the present study, we evaluated the plasma pharmacokinetic profile of tectoridin in rats. We isolated a new metabolite, tectorigenin-7-O-glucuronide-4′-O-sulfate (Te-7G-4′S), from the bile of rats treated orally with tectoridin and determined its chemical structure by spectral analysis. Furthermore, we developed a selective and accurate method for the simultaneous quantification of tectoridin metabolites, including Te-7G-4′S, tectorigenin-7-O-glucuronide (Te-7G), tectorigenin-7-O-sulfate (Te-7S), and tectorigenin in rat plasma, and measured their plasma concentrations in rats orally administered tectoridin (200 mg/kg). Plasma concentrations of Te-7G-4′S, Te-7G, Te-7S, and tectorigenin reached maximal values of 21.4 ± 13.8 μmol at 3.50 ± 1.87 h, 20.5 ± 9.7 μmol at 3.17 ± 1.81 h, 14.3 ± 3.3 μmol at 5.58 ± 3.07 h, and 8.67 ± 3.07 μmol at 4.92 ± 2.87 h, respectively. Enterohepatic recirculation resulted in double peaks or a flat concentration curve/time profile of the metabolites. Since plasma concentrations of tectorigenin conjugated metabolites were higher than those of the tectorigenin aglycone, it can be concluded that extensive phase II metabolism plays an important role in the pharmacokinetics of tectoridin and tectorigenin in vivo.
Keywords: Tectoridin; Plasma pharmacokinetic; Glucuronidation; Sulfation; Rat;

► Sequential native fluorescence–ESI-MS quantification of HPLC-separated polypeptides is enabled. ► Peptides and proteins containing all natively fluorescent amino acids are quantified. ► Figures of merit for spectrofluorimetric and ESI-MS quantifications of natively fluorescent biomolecules are complementary. ► Native fluorescence provides better linearity and repeatability of quantification than ESI-MS. ► This is a label-free technique, which can facilitate quantification of peptides and proteins by LC–ESI-MS.Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS.
Keywords: Quantification; Peptides; Proteins; HPLC; Native fluorescence; ESI-MS;

Diffusion Split-Flow Thin Cell (SPLITT) system for protein separations by Srinivas Merugu; Himanshu J. Sant; Bruce K. Gale (78-83).
► Continuous removal of beta-2-microglobulin and parathyroid hormone. ► COMSOL multiphysics model of diffusion SPLITT system. ► The model is used to optimize experimental parameters. ► The technique has the potential to be used as an additional step in traditional dialysis protocols.A diffusion Split-Flow Thin Cell (SPLITT) system was used to partially remove small peptides such as β2 microglobulin (β2M) and parathyroid hormone (PTH) in a continuous manner from an input flow stream while preserving most (over 97%) of the larger protein in the sample, such as albumin. To help determine the operating conditions for this work, a two-dimensional numerical model based on the Navier–Stokes equation and convection–diffusion equations was developed for diffusional SPLITT using COMSOL multiphysics software (COMSOL Inc., MA). These simulations were used to obtain the relationship between important operational parameters and the purification efficiency for proteins of interest. The diffusion-based SPLITT system was fabricated using xurography and was used to demonstrate protein purification based on the differences in size or diffusion coefficient of the sample. The results obtained from the experiments are compared with the mathematical model and show good agreement, while the variations between these results are discussed. The results show that significant portions of small peptides (>25%) can be removed while preserving larger proteins (up to 95%) in the carrier stream. A potential application of this technique is to be used as an additional step in kidney dialysis to remove toxins that are not effectively removed by current dialysis protocols.
Keywords: Field flow fractionation; Microfluidics; Split-Flow Thin Cell; Dialysis; Continuous separation;

Validation of a sequential extraction and liquid chromatography–tandem mass spectrometric method for determination of dihydrotestosterone, androstanediol and androstanediol–glucuronide in prostate tissues by Hans H. Adomat; Onkar S. Bains; Joanna M. Lubieniecka; Martin E. Gleave; Emma S. Guns; Thomas A. Grigliatti; Ronald E. Reid; K. Wayne Riggs (84-95).
► Sensitive method for tissue analysis of hydroxyl containing steroids. ► Extraction permitting derivatization of steroids and analysis of glucuronides. ► Optimized UPLC eliminates post derivatizing SPE requirements. ► Optional capability to separate α and β isomers with chiral column.Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17β-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50 mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.
Keywords: Dihydrotestosterone; Androstanediol; Androstanediol–glucuronide; LC–MS/MS; Fluoromethylpyridinium p-toluenesulfonate derivatization; Sequential extraction;

► Eleven antihypertensives were simultaneously assayed in isocratic mode within 10 min. ► The proposed method enables a comprehensive study in three most concerned systems. ► The method provides a more general condition for analyses in various systems.A novel chemometric-assisted high performance liquid chromatography method coupled with diode array detector (HPLC-DAD) was presented for the simultaneous determination of eleven antihypertensives from multiple classes in most concerned matrix systems. With the aid of second-order calibration which enables specific information of analytes to be well extracted, the heavily overlapping profiles between analytes and the coeluting interferences can be successfully separated and thus accurately quantified. A great advantage of the novel strategy lies in the fact that the analysis could be carried out with the same isocratic mobile phase (methanol/KH2PO4: 58:42, v/v, pH 2.60) in a short time regardless of the changes of matrices, such as human serum, health product and Chinese patent medicine. Both qualitative and quantitative results indicate that the hybrid strategy that using HPLC-DAD coupled with second-order chemometric method would be a high performance approach for the purpose of simultaneously quantifying multiple classes of antihypertensives in complex systems. Additionally, the analytical strategy can potentially benefit drug monitoring in both therapeutic research and pharmaceutical quality control. Moreover, the accuracy and reliability of the proposed methodology has been evaluated using several statistical parameters such as root mean squared error of prediction (RMSEP), figures of merit (FOM) and reproducibility of inter-day analysis.
Keywords: Second-order calibration; High performance liquid chromatography; Antihypertensives; Human serum; Health product; Chinese patent medicine;

► A novel IMAC material for phosphopeptides capture was prepared based on cellulose. ► 14 phosphopeptides were identified from 2 pmol α-casein lysates. ► 15 phosphopeptides were identified from α-casein and BSA digests mixture (1:100). ► The material was applied successfully in rice phosphoproteome analysis.In this study, a novel immobilized metal ion affinity chromatography (IMAC) material for phosphopeptide enrichment was prepared based on modified cellulose and was applied in rice phosphoproteome analysis. Firstly, cellulose was modified with phosphoric acid via esterification, and then Ti4+ was chelated onto the phosphorylated cellulose. The synthesized materials were ultrafine powders and had good dispersibility in acidic buffer, and as supporting matrix, phosphorylated cellulose exhibited good biocompatibility and chemical stability. Enrichment conditions were optimized and the optimum loading buffer was 40% acetonitrile (ACN) with 6% trifluoroacetic acid (TFA). Finally, the Ti4+-phosphate functionalized cellulose was submitted to phosphopeptides enrichment prior to mass spectrometry (MS). For α-casein lysates, 14 phosphopeptides were detected with high intensities even though the sample concentration was as low as 2 pmol. Besides, 15 phosphopeptides were still identified by using the digest mixture of α-casein and bovine serum albumin with molar ratio of 1:100, which demonstrated high specificity and sensitivity for phosphopeptides enrichment. 19 phosphoproteins were identified from 200 μg of salt-free rice leaf protein lysates, while 30 phosphoproteins were identified from salt-stressed rice leaf protein lysates, and most of these proteins were related to the biological processes in response to abiotic stimulus.
Keywords: Phosphorylated cellulose; Immobilized metal ion affinity chromatography; Mass spectrometry; Phosphopeptides enrichment; Rice phosphoproteome;

► LC–MS/MS method for simultaneous quantitation of oxymorphone and 6-OH-oxymorphone. ► Low LOQ for oxymorphone (35 pg/mL) and 6-OH-oxymorphone (25 pg/mL) was achieved. ► Optimization of chromatographic separation and sample extraction. ► Elimination of the possible interference resulting from in-source conversion.A selective high performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the simultaneous determination of oxymorphone and its active metabolite 6-OH-oxymorphone in human plasma was developed and validated using oxymorphone-d3 as the internal standard. Chromatographic conditions were optimized to separate oxymorphone from the other metabolite, oxymorphone-3-glucuronide, which may convert to oxymorphone in MS ion source, resulting in inaccurate quantitation of oxymorphone. Solid phase extraction (SPE) was used to extract oxymorphone and 6-OH-oxymorphone from plasma. SPE offered the advantage of being able to remove the unwanted metabolite, oxymorphone-3-glucuronide, through the wash step during the extraction. The developed method was precise and reproducible as shown by good linearity of calibration curves (correlation coefficients ≥0.9968 for oxymorphone and ≥0.9967 for 6-OH-oxymorphone) with high intraday assay and interday assay precision (CV% ≤11.0% for oxymorphone and ≤12.6% for 6-OH-oxymorphone) over a range of 35/25 – 5000/5000 pg/mL for oxymorphone/6-OH-oxymorphone. The method has been successfully applied to analyze oxymorphone and 6-OH-oxymorphone in plasma from 19 healthy volunteers in a bioequivalence study. A total of 1026 samples were analyzed. Good linearity (average correlation coefficient 0.9988 for oxymorphone and 0.9966 for 6-OH-oxymorphone) was achieved with calibration curves and high precision (CV% ≤5.9% for oxymorphone and ≤10.9% for 6-OH-oxymorphone) was obtained with QCs.
Keywords: Oxymorphone; 6-OH-oxymorphone; LC–MS/MS; SPE; Human plasma;

Challenges in the simultaneous quantitation of sumatriptan and naproxen in human plasma: Application to a bioequivalence study by Daxesh P. Patel; Primal Sharma; Mallika Sanyal; Puran Singhal; Pranav S. Shrivastav (122-131).
► First UPLC–MS/MS method for simultaneous determination of sumatriptan and naproxen. ► Highly sensitive and rapid compared to all existing methods in biological matrices. ► Matrix effect was evaluated by qualitative and quantitative assessment. ► Successful bioequivalence study in healthy Indian subjects for a fixed dose formulation. ► Reproducibility of study data is demonstrated by incurred sample reanalysis.An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the simultaneous determination of sumatriptan and naproxen in human plasma using naratriptan and indomethacin as the internal standards (ISs). The plasma samples were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample. Chromatography was carried out on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column under isocratic conditions using a mobile phase consisting of methanol–acetonitrile–4.0 mM ammonium acetate (70:10:20, v/v/v). The precursor → product ion transition for both the analytes and ISs was monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a wide dynamic concentration range of 0.050–100 ng/mL for sumatriptan and 0.050–100 μg/mL for naproxen. Matrix effect was assessed by post-column analyte infusion and the extraction recovery was >95.0% across four quality control levels for both the analytes. Stability was evaluated under different conditions including bench top, processed sample, freeze and thaw and long term. The method was applied to support a bioequivalence study of 85 mg sumatriptan + 500 mg naproxen sodium fixed dose formulation in 28 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 123 incurred samples.
Keywords: Sumatriptan; Naproxen; UPLC–MS/MS; Sensitive; High throughput; Bioequivalence;

Metaxalone estimation in biological matrix using high-throughput LC–MS/MS bioanalytical method by Dipanjan Goswami; Arabinda Saha; Sanjay Gurule; Arshad Khuroo; Tausif Monif; Poonam Vats (132-136).
► High-throughput bioanalytical method was developed and validated adhering to global regulatory norms. ► We examined metaxalone in blood, plasma and in hemolyzed/lipemic plasma for effect of matrix component interference. ► Achieved mean recovery >78% for both metaxalone and its internal standard and intra- and inter-day precisions and accuracy were all within 6%. ► We have found that matrix effect was negligible though the method is sensitive and demonstrated high extraction efficiency.Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytical methods lacked detailed stability evaluation in blood and plasma. An accurate, precise, high-throughput tandem mass spectroscopic method has been developed and validated. Following solid phase extraction (SPE), metaxalone and the internal standard metaxalone-d3 were extracted from an aliquot of 200 μL of human plasma. Chromatographic separation achieved on an Ascentis Express C18 column (50 mm × 4.6 mm i.d., 2.7 μm particle size) with mobile phase is a mixture of 10 mM ammonium acetate buffer (pH 4.5)–methanol–acetonitrile (20:50:30, v/v/v), at an isocratic flow rate of 0.7 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The mass transitions of metaxalone and metaxalone-d3 were m/z 222.3 → 161.2 and m/z 225.3 → 163.3, respectively. The linear calibration curves were obtained in the concentration range of 0.105–10.081 μg/mL (r 2  ≥ 0.99) with a lower limit of quantification (LLOQ) of 0.105 μg/mL. The intra- and inter-day precisions and relative error were all within 6%. Despite achieving high mean recovery (>78%), no interference peaks or matrix effects were observed. Detailed stability exercises including drug stability in blood, hemolyzed, lipemic and normal plasma were conducted to extend the method applicability in vast majority of clinical studies using 800 mg metaxalone extended release oral dosage form.
Keywords: Metaxalone; LC–MS/MS; Method development; Matrix stability; Method validation;

► Simple and rapid method to determine celecoxib in human plasma with a total running time of 2 min for each sample. ► Validated for specificity, LLOQ, recovery, accuracy, precision, stability and linearity. ► Applied to analyze more than 1200 clinical samples by using the proposed method.A suitable liquid chromatography tandem mass spectrometry (LC–MS/MS) method to determine celecoxib in human plasma is needed for bioequivalence and pharmacokinetic studies of celecoxib preparations. The present study describes a simple, rapid, reproducible, and reliable LC–MS/MS method to determine celecoxib concentrations in human plasma. After one-step liquid–liquid extraction (LLE) using methyl tert-butyl ether (MTBE), celecoxib and atorvastatin (internal standard, IS) were eluted on a Luna HILIC column with an isocratic mobile phase, consisting of 10 mM ammonium formate buffer (adjusted to pH 3.0 with formic acid):methanol (5:95, v/v) at a flow rate of 0.2 mL/min. The achieved lower limit of quantitation (LLOQ) was 10 ng/mL (S/N > 10) and the standard calibration curve for celecoxib was linear (correlation coefficients were >0.9995) over the studied concentration range (10–2000 ng/mL). The inter- and intra-assay coefficients of variation ranged from 1.15% to 4.93% and 1.08% to 7.81%, respectively. The chromatographic run time for each plasma sample was <2 min. The developed method was successfully applied to a bioequivalence study of celecoxib in healthy Korean male volunteers.
Keywords: Celecoxib; LC–MS/MS; Liquid–liquid extraction; Bioequivalence;

Simultaneous determination of primaquine and carboxyprimaquine in plasma using solid phase extraction and LC–MS assay by Madhu Page-Sharp; Kenneth F. Ilett; Inoni Betuela; Timothy M.E. Davis; Kevin T. Batty (142-146).
► We report a LC–MS assay for primaquine and its active metabolite, carboxyprimaquine. ► A simultaneous, solid phase extraction from human plasma was developed. ► Limit of quantification was 2 μg/L for primaquine and 2.5 μg/L for carboxyprimaquine. ► Run time was <10 min and recovery >85%. ► Application was shown using plasma samples from children who were given primaquine.Sensitive bioanalytical methods are required for pharmacokinetic studies in children, due to the small volume and modest number of samples that can be obtained. We sought to develop a LC–MS assay for primaquine and its active metabolite, carboxyprimaquine, following simultaneous, solid phase extraction of both analytes from human plasma. The analysis was conducted on a single-quad LC–MS system (Shimadzu Model 2020) in ESI+ mode, with quantitation by selected ion monitoring. Primaquine, carboxyprimaquine and 8-aminoquinoline (internal standard) were separated using a mobile phase of 80:20 methanol:water with 0.1% (v/v) formic acid and a Luna C18 HPLC column, at ambient temperature. Solid phase extraction of the analytes from plasma (0.5 mL) was achieved with Oasis® HLB cartridges. The retention times for primaquine, 8-aminoquinoline and carboxyprimaquine were 3.3, 5.7 and 8.5 min, respectively. The calibration curve range (2–1500 μg/L) was appropriate for the limits of quantification and detection for primaquine (2 μg/L and 1 μg/L, respectively) and carboxyprimaquine (2.5 μg/L and 1 μg/L) and the anticipated plasma concentrations of the analytes. Intra- and inter-day precision for both primaquine and carboxyprimaquine was <10% across the concentration range 5–1000 μg/L. Accuracy for both analytes was <15% (5–500 μg/L). This validated LC–MS method with solid phase extraction facilitates the simultaneous analysis of primaquine and carboxyprimaquine from small volumes of human plasma, with run time <10 min, recovery >85% and sensitivity of 1–2 μg/L.
Keywords: Primaquine; Carboxyprimaquine; Plasma; Solid phase extraction; LC–MS;

Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping by Yuri E.M. van der Burgt; Christa M. Cobbaert; Hans Dalebout; Nico Smit; André M. Deelder (147-150).
► Stability of troponin monitored with MS-based peptide mapping approach. ► Use of the “semiTrypsin” option in Mascot to find unexpected peptides. ► Results provide novel data on instability of the troponin standard from NIST.In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC–MS/MS. From a Mascot database search in combination with “semiTrypsin” tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37 °C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of “unexpected” peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a “single-step” MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed.
Keywords: Cardiac troponin; Peptide mapping; Stability;

The bioanalysis of the major Echinacea purpurea constituents dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides in human plasma using LC–MS/MS by Andrew K.L. Goey; Hilde Rosing; Irma Meijerman; Rolf W. Sparidans; Jan H.M. Schellens; Jos H. Beijnen (151-156).
► An LC–MS/MS assay for the most abundant alkylamides (DTAI) in E. purpurea has been validated for quantification in human plasma. ► Most sensitive FDA-validated bioanalytical assay for DTAI with a quantification limit of 0.01 ng/mL. ► Only 300 μL plasma underwent simple liquid–liquid extraction. Run time was 7.5 min. ► DTAI was successfully quantified in patients after ingestion of E. purpurea extract.Alkylamides are a group of active components of the widely used herb Echinacea purpurea (E. purpurea), which have immunostimulatory and anti-inflammatory effects. For the most abundant alkylamides, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI), an LC–MS/MS assay has been developed and validated for quantification in human plasma. This assay will be used to support a clinical interaction study with E. purpurea. A 300 μL plasma aliquot underwent liquid–liquid extraction with diethylether-n-hexane (50:50, v/v). After evaporization and reconstitution in 100 μL of acetonitrile–water (50:50, v/v) 20 μL of sample were injected into the HPLC system. Chromatographic separation was achieved with a Polaris 3 C18-A column (50 mm × 2 mm ID, particle size 3 μm), a flow rate of 0.3 mL/min and isocratic elution with acetonitrile–water (50:50, v/v) containing 0.1% formic acid during the first 5 min. Hereafter, gradient elution was applied for 0.5 min, followed by restoration of the initial isocratic conditions. The total run time was 7.5 min. The assay was validated over a concentration range from 0.01 to 50 ng/mL for DTAI, with a lower limit of quantification of 0.01 ng/mL. Validation results show that DTAI can be accurately and precisely quantified in human plasma. DTAI also demonstrated to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated by measuring the plasma concentration of DTAI in patients after ingestion of a commercial extract of E. purpurea.
Keywords: Dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides; Echinacea purpurea; Alkylamide; LC–MS/MS; Human plasma;