Journal of Chromatography B (v.893-894, #C)

► The use of internal standards for protein analysis with LC–MS is reviewed. ► A categorization is made into protein-based and peptide-based internal standards. ► The ability to correct for analytical variability is compared and discussed. ► There is no clearly preferable type of internal standardization for proteins.Following the increase in development of protein biopharmaceuticals, there is a growing demand for the sensitive and reliable quantification of these proteins in complex biological matrices such as plasma and serum to support (pre)-clinical research. In this field, ligand binding assays (LBAs) are currently the standard analytical technique, but in recent years, there is a trend towards the use of liquid chromatography hyphenated with (tandem) mass spectrometry (LC–MS/MS). One of the reasons for this trend is the possibility to use internal standards to correct for analytical variability and thus improve the precision and accuracy of the results. In the LC–MS/MS bioanalysis of small molecules, internal standardization is quite straightforward: either a stable-isotope labeled (SIL) form of the analyte or a structural analogue is used. For the quantification of biopharmaceutical proteins, the situation is more complex. Since the protein of interest is digested to a mixture of peptides, one of which is subsequently used for quantification, there are more options for internal standardization. A SIL form or a structural analogue of either the intact protein or the signature peptide can be used. In addition, a modified form of the SIL-peptide internal standard, containing one or more cleavable groups is a possibility, and an internal standard can be generated during the analysis by using differential derivatization techniques. In this paper we provide an overview of the different options for internal standardization in the field of absolute targeted quantification of protein biopharmaceuticals using LC–MS/MS, based on literature from 2003 to 2011. The advantages and disadvantages of the different approaches are evaluated both with regard to the correction they provide for the variability of the different steps of the analysis and with regard to their generic availability. As most of the approaches used lead to acceptable results in terms of accuracy and precision, we conclude that there currently is no clear preferable method for internal standardization in the field of protein quantification by LC–MS/MS. It is essential, however, that any step in the analysis that is not covered by the internal standard chosen, should be carefully optimized and controlled.
Keywords: Internal standards; Bioanalysis; Biopharmaceuticals; Protein quantification; Quantification; LC–MS/MS;

Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry by Sai P. Bathena; Jiangeng Huang; Adrian A. Epstein; Howard E. Gendelman; Michael D. Boska; Yazen Alnouti (15-20).
► A sensitive, selective, and simple LC–MS/MS method using HILIC was validated for the quantification of nine AAs and MI in mouse brain. ► One step methanol–protein precipitation was used for sample extraction. ► Method validation was performed in the range of 2.5–20 to 500–2000 ng/ml to ensure the simultaneous quantification of all analytes of interest.Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5–20 to 500–4000 ng/ml. This LC–MS/MS method was validated for biomarker discovery in models of human neurological disorders.
Keywords: LC–MS/MS; Amino acids; Myo-inositol; HILIC; Multiple reaction monitoring (MRM);

► Fully automated on-line SPE-HPLC utilized for PK evaluation of bavachinin. ► On-line SPE removed the time-consuming, tedious and costly manual process. ► The method is simple, fast, specific and sensitive.A fully automated on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (SPE cartridge and analytical column) connected via a Valco 6-port switching valve. Plasma samples (10 μL) were injected directly onto a C18 SPE cartridge (MF Ph-1 C18, 10 mm × 4 mm, 5 μm) and the biological matrix was washed out for 2 min with the loading solvent (5 mM NaH2PO4 buffer, pH 3.5) at a flow rate of 1 mL/min. By rotation of the switching valve, bavachinin was eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column (Venusil MP C18, 4.6 mm × 150 mm, 5 μm) by the chromatographic mobile phase consisted of acetonitrile-5 mM NaH2PO4 buffer 65/35 (v/v, pH 3.5) at a flow rate of 1 mL/min. The complete cycle of the on-line SPE purification and chromatographic separation of the analyte was 13 min with UV detection performed at 236 nm. Calibration curve with good linearity (r  = 0.9997) was obtained in the range of 20–4000 ng/mL in mouse plasma. The intra-day and inter-day precisions (RSD) of bavachinin were in the range of 0.20–2.32% and the accuracies were between 98.47% and 102.95%. The lower limit of quantification (LLOQ) of the assay was 20 ng/mL. In conclusion, the established automated on-line SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy, and was successfully utilized to quantify bavachinin in mouse plasma to support the pharmacokinetic (PK) studies. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.
Keywords: Bavachinin; On-line solid-phase extraction; HPLC; Pharmacokinetic study;

► WJ-38 is a potent aldose reductase inhibitor for diabetic complications. ► It is a strongly protein-bound compound (over 97% was bound to plasma proteins). ► A sensitive and specific LC–MS/MS method was developed for WJ-38 analysis. ► Special emphasis was focused on the effective extraction of WJ-38 from rat plasma. ► The chromatographic run time was 3.5 min and the LLOQ was 10.0 ng/mL.WJ-38 is an aldose reductase inhibitor that is being developed for the treatment of diabetic complications. The present paper describes a sensitive and specific liquid chromatography-tandem mass spectrometry method for the determination of WJ-38 in rat plasma. Partial denaturation of plasma proteins with methanol followed by liquid–liquid extraction using ethyl acetate was used to extract strongly protein-bound WJ-38 from rat plasma. Chromatographic separation was performed on an Inertsil ODS-3 column with an isocratic mobile phase consisting of acetonitrile, water and formic acid (75:25:0.125, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. Quantitation was performed using selected reaction monitoring of precursor-product ion transitions at m/z 392 → 246 for WJ-38 and m/z 446 → 321 for glipizide (internal standard). A linear calibration curve was obtained over the concentration range of 10.0–10,000 ng/mL for WJ-38 in rat plasma. The intra- and inter-day precisions were less than 13.6% and the accuracy was within ±5.3%. The extraction recovery of WJ-38 from rat plasma was over 66.0%. The validated method has been successfully applied to a pharmacokinetic study in rats after intragastrical administration of WJ-38.
Keywords: Aldose reductase inhibitor; WJ-38; Liquid chromatography-tandem mass spectrometry; Rat plasma; Pharmacokinetics;

LC/LC–MS/MS of an innovative prostate human epithelial cancer (PHEC) in vitro model system by John D. Lapek; James L. McGrath; William A. Ricke; Alan E. Friedman (34-42).
► Proteomic characterization of a novel in vitro prostate cancer model system. ► Progressive cancer states in vivo: non-tumorigenic, tumorigenic, and metastatic. ► Differential protein expression using PF2D followed by mass spectral identification.This work describes the proteomic characterization of a novel in vitro prostate cancer model system, the clonal prostatic human epithelial cancer (PHEC) cell lines. The model is composed of three cell lines representing the three progressive cancer states found in vivo: non-tumorigenic, tumorigenic, and metastatic. The cell lines were evaluated for differential protein expression between states using two dimensional liquid:liquid chromatographic separation followed by mass spectral identification. The proteins from cellular extracts were first separated using liquid:liquid primary separation based on their isoelectric points and hydrophobicity. The resulting peptide fractions were applied to liquid chromatography–mass spectrometry (LC–MS) separation for mass determination and protein identification based on Mascot database inquiry. Over 200 proteins that change expression over the course of progression of this in vitro prostate cancer model were discovered during the comparative analysis of the three cell lines. The importance of these proteins on prostate cancer progression remains to be elucidated with further characterizations. The combination of the two dimensional liquid:liquid separation and mass spectral identifications was used to successfully analyze differential protein expression between multiple cell lines.
Keywords: PF2D; Prostate cancer progression;

Enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino by column chromatography technique by Jian-Fang Chen; Gui-Ming Fu; Yin Wan; Cheng-Mei Liu; Jian-Xin Chai; Hong-Ge Li; Jian-Tao Wang; Ming-Hu; Lun-Ning Zhang (43-48).
► The performance of resins for the purification of gardenia yellow was evaluated. ► HPD722 resin was chosen and its condition for purification was optimized. ► Under the optimal condition, the colority of the product obtained were up to 300.In present study, the performance and separation characteristics of nine macroporous resins for the enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino have been evaluated. The adsorption and desorption properties of crude gardenia yellow solution on macroporous resins including HPD722, HPD100, HPD100A, HPD400, HPD400A, D101, AB-8, XAD-16, and NKA-9 have been compared. Then, HPD722 was chosen to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. Column packed with HPD722 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of gardenia yellow. The optimal conditions were as follows: The crude gardenia yellow solution with concentration of 15 mg/mL was loaded in column packed with HPD722 resin at the flow rate of 1.0 mL/min, and the adsorbate-laden column was washed with 800 mL water, 600 mL 15% ethanol water solution respectively at the speed of 2.5 mL/min, then desorbed with 200 mL 80% ethanol water solution at the speed of 3.5 mL/min. The colority of the product obtained were up to 300. The method developed in this study provides a new approach for scale-up separation and purification of gardenia yellow from G. jasminoides var. radicans Makino.
Keywords: Gardenia yellow; Adsorption; Desorption; Macroporous resin; Colority;

► A derivatization LC–MS/MS method for quantitation of pyrimidines was established. ► Three derivatization reagents were carefully compared. ► Three derivatization modes were tried. ► The matrix effect and interferences were eliminated by the gradient elution.A comparison of three derivatization reagents (dansyl chloride, diazomethane and p-bromophenacyl bromide) for the simultaneous quantitation of three anticancer chemicals (tegafur, 5-fluorouracil and gimeracil) and endogenous uracil in plasma using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and evaluated. Through a comprehensive consideration, p-bromophenacyl bromide (p-BPB) was finally selected as the derivatization reagent. Because it essentially changed the chromatographic behavior of the aforementioned highly hydrophilic compounds and significantly enhanced their sensitivities. The method was validated over the concentration ranges of 5–5000 ng/ml for tegafur, 0.6–700 ng/ml for 5-fluorouracil, 3–700 ng/ml for gimeracil and 6–2000 ng/ml for uracil. The method was successfully applied to the pharmacokinetics study of tegafur, 5-fluorouracil, gimeracil and uracil in cancer patients.
Keywords: Derivatization; Tegafur; 5-Fluorouracil; Gimeracil; Uracil; LC–MS/MS; Pharmacokinetics;

► Analysis of estradiol and estrone by LC–MS/MS without derisvatization. ► LOQ below 0.5 pg/mL for estradiol and estrone. ► Suitable for analysis in children and post-menopausal women.Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC–MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2 mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.
Keywords: Estrone; Estradiol; Estrogens; Tandem mass spectrometry;

Semi-automated solid-phase extraction method for studying the biodegradation of ochratoxin A by human intestinal microbiota by Valérie Camel; Minale Ouethrani; Cindy Coudray; Catherine Philippe; Sylvie Rabot (63-68).
► A simple and rapid semi-automated SPE method for the analysis of ochratoxin A in digestive contents and faecal excreta. ► Suitability of the method for the analysis of ochratoxin B in faecal excreta. ► Preliminary results of ochratoxin A biodegradation studies by the human intestinal microbiota under simple in vitro conditions. ► Partial biodegradation of ochratoxin A, and identification of three phase I metabolites (ochratoxin α, ochratoxin B and open ochratoxin A).A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9 mL of air), the cartridge was rinsed with 5-mL H2O/ACN (80:20, v/v), before eluting the compounds with 3× 1 mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled; this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72 h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin α, open ochratoxin A and ochratoxin B.
Keywords: Biodegradation; Human intestinal microbiota; Ochratoxin A; Solid-phase extraction;

► Cibacron Blue F3GA attached supermacroporous cryogels for human interferon adsorption. ► FPLC system was used for interferon purification from human gingival fibroblast extract. ► The purified interferon samples have 97.6% purity determined by SDS–PAGE. ► After repeated tenth use, no significant decrease was determined in the adsorption capacity. ► The cryogels are potential candidate for rapid, cheap and specific interferon purification.In this study, we have focused our attention on preparing supermacroporous cryogels as a potential dye-affinity adsorbent for interferon purification. For this purpose, 2-hydroxyethyl methacrylate (HEMA) and Cibacron Blue F3GA (CB) were selected as main monomer and dye–ligand. Cibacron Blue F3GA attached supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)/CB] cryogels were prepared and characterized by swelling test, scanning electron microscopy, elemental analysis, and FTIR. After that, the effecting factors such as pH, concentration, interaction time, and ionic strength on the interferon separation were evaluated. The maximum adsorption capacity of poly(HEMA)/CB cryogels was obtained as 38.2 mg/g at pH 6.0. Fast protein liquid chromatography (FPLC) system was used for interferon purification from human gingival fibroblast extract. The chromatography parameters, capacity and selectivity factors, resolution and theoretical plate number were found as 7.79, 9.62, 4.23 and 554, respectively. Although some decreases in total protein content, from 320 μg to 18 μg, and interferon activity, from 2.6 × 103  IU to 2.2 × 103  IU, were determined, specific antiviral activity increased from 7.19 IU/μg to 122.2 IU/μg. The purified interferon samples have 97.6% purity determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After repeated ten adsorption–desorption cycles, no significant decrease was determined in adsorption capacity of cryogel. In result, poly(HEMA)/CB cryogels have an application potential for rapid, cheap and specific purification of interferon.
Keywords: Poly(HEMA) cryogel; Cibacron Blue F3GA; Interferon; Affinity purification; Fast protein liquid chromatography;

HPLC–DAD protein kinase inhibitor analysis in human serum by Marek Dziadosz; Rüdiger Lessig; Heidemarie Bartels (77-81).
► An HPLC–DAD method for protein kinase inhibitor analysis was validated. ► A comparison with LC/MS/MS is described. ► The advantage over other methods/techniques is discussed.We here describe an HPLC–DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib – internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H3PO4/KH2PO4 (pH = 2.3)–acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid–liquid extraction led to good recoveries (73–90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20–10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1 mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range.
Keywords: Vatalanib; Bosutinib; Canertinib; Tandutinib; Pazopanib; Erlotinib;

► Quantification of roflumilast and its metabolite, a PDE inhibitor against COPD in human plasma. ► High throughput bioanalytical method with liquid extraction and LC–MS/MS. ► Parallel chromatography in a dual column-switching mode. ► Positive ion selected reaction monitoring with pneumatically assisted ESI.A high throughput bioanalytical method based on semi-automated liquid extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the sensitive quantification of roflumilast and its metabolite roflumilast N-oxide, a phosphodiesterase (PDE) inhibitor in human plasma and serum. The sample work-up procedure comprised liquid extraction using penta-deuterated analogues of both analytes as internal standards. Chromatography was performed on C18 revered phase analytical columns at a flow rate of 0.5 mL/min in the dual column mode employing a column switching technique and a linear gradient from 18% to 54% acetonitrile in 0.005 M aqueous ammonium acetate containing 0.006% formic acid. Mass spectrometry was performed on an API 4000 instrument in the positive ion SRM-mode (selected reaction monitoring) with the Turbo-V® ionspray interface. The method showed linear detector responses over the entire calibration range between 0.1 ng/mL (lower limit of quantification (LLOQ)) and 50 ng/mL (upper limit of quantification (ULOQ)) for both analytes. Linear regression analysis with concentration-squared weighting (1/x 2 for roflumilast and 1/x for roflumilast N-oxide) yielded inaccuracy and precision values <15% and coefficients of correlation (r) for the calibration curves >0.99 for both analytes.
Keywords: Daxas; Roflumilast; Parallel chromatography; Tandem mass spectrometry; ESI; LC–MS/MS;

Development and validation of LC–MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine by A.C. Dubbelman; M. Tibben; H. Rosing; A. Gebretensae; L. Nan; S.H. Gorman; P. Robertson; J.H.M. Schellens; J.H. Beijnen (92-100).
► Bendamustine is an alkylating agent, used for haematological malignancies. ► Quantification of bendamustine is difficult due to chemical degradation. ► We developed quantitative assays for bendamustine, minimizing its degradation. ► The assays quantify bendamustine and metabolites in human plasma and urine. ► The assays were validated and successfully applied to clinical samples.A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine.Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200 μL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5 mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer.The quantifiable range for bendamustine, M3 and M4 was 0.5–500 ng/mL in plasma and 0.5–50 μg/mL in urine, and that for HP2 was 1–500 ng/mL in plasma and 0.1–50 μg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ±20% of nominal and CV values below 20% at the lower limit of quantification and within ±15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.
Keywords: Bendamustine; Metabolites; Alkylating agent; Stability; LC–MS/MS;

► We have developed a SPE/P-HPLC system to purify chrysophanol in large quantities. ► The recovery of chrysophanol in the system was in the range of 88–91.5%. ► The purity of the isolated chrysophanol was as high as 99%. ► The SFE/P-HPLC system can isolate non-polar compounds for safer medicines.Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25× higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210 atm and 85 °C for 30 min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10 mL/min. According to 1H NMR and LC–MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88–91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38 mg/g) and the shortest run time (3 h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future.
Keywords: Chrysophanol; Supercritical fluid extraction; Preparative liquid chromatography; Rheum Palmatum LINN;

Serum metabolomic profiles from patients with acute kidney injury: A pilot study by Jinchun Sun; Melissa Shannon; Yosuke Ando; Laura K. Schnackenberg; Nasim A. Khan; Didier Portilla; Richard D. Beger (107-113).
► LC/MS-based metabolic profiling of serum discovered novel indicators of AKI. ► Homocysteine, pyroglutamate and dimethylarginine (ADMA) increased in AKI patients. ► Homocysteine and ADMA are good indicators of AKI. ► Increases in acylcarnitines indicated fatty acid oxidation inhibited in AKI patients.Low sensitivity of current clinical markers (serum creatinine and blood urea nitrogen (BUN)) in early stages of the development of acute kidney injury (AKI) limits their utility. Rapid LC/MS-based metabolic profiling of serum demonstrated in a pilot study that metabolomics could provide novel indicators of AKI. Metabolic profiles of serum samples from seventeen hospitalized patients with newly diagnosed AKI were compared with the profiles of serum from age-matched subjects with normal kidney function. Increases in acylcarnitines and amino acids (methionine, homocysteine, pyroglutamate, asymmetric dimethylarginine (ADMA), and phenylalanine) and a reduction in serum levels of arginine and several lysophosphatidyl cholines were observed in patients with AKI compared to healthy subjects. Increases in homocysteine, ADMA and pyroglutamate have been recognized as biomarkers of cardiovascular and renal disease, and acylcarnitines represent biomarkers of defective fatty acid oxidation. The results of this pilot study demonstrate the utility of metabolomics in the discovery of novel serum biomarkers that can facilitate the diagnosis and determine prognosis of AKI in hospitalized patients.
Keywords: Acute kidney injury; Metabolic profiling; LC/MS; Serum biomarkers; Acylcarnitines;

► A novel HILIC/ESI-MS method to quantify deferasirox in human plasma is proposed. ► Retention mechanisms of the analytes on XBridge®-HILIC column have been investigated. ► The method was applied to the analysis clinical samples of β-thalassemia patients.A rapid hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed, validated and applied to the determination of deferasirox, in human plasma. The sample preparation process involved liquid–liquid extraction of 50 μL plasma sample using ethyl acetate as an extraction solvent. Chromatographic separation was performed on an XBridge®-HILIC analytical column (150.0 mm × 2.1 mm i.d., particle size 3.5 μm, 135 Å) under isocratic elution. The mobile phase was composed of a 10% 8.0 mM ammonium acetate water solution pH = 5.0, adjusted with formic acid, in a binary mixture of acetonitrile/methanol (50:50, v/v) and pumped at a flow rate of 0.20 mL/min. Quantitation of deferasirox was performed with selected ion monitoring (SIM) in positive ionization mode using electrospray ionization interface. The assay was found to be linear in the concentration range of 0.20–120.0 μg/mL for deferasirox. Intermediate precision was found less than 3.9% over the tested concentration ranges. A run time of less than 6.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to support a wide range of clinical studies concerning deferasirox monitoring and it was applied to the analysis of human plasma samples obtained from patients with β-thalassemia major.
Keywords: Hydrophilic interaction liquid chromatography (HILIC); Liquid chromatography/mass spectrometry; Deferasirox; Mirtazapine; Human plasma;

Separation and purification of phosvitin phosphopeptides using immobilized metal affinity nanoparticles by Jing Zhang; Jun Sun; Yuntao Liu; Junhua Li; Yujie Su; Wenshui Xia; Yanjun Yang (121-126).
► PPPs were purified directly from crude egg yolk hydrolysis polypeptides. ► High-efficiency and low-cost Fe3O4 (PEG + CS) @ Fe (III) nanoparticles have been developed. ► The important functional groups were identified by Fourier Transform Infrared technique. ► The characteristics of the immobilized metal affinity nanoparticles were discussed.Monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as IMAN @ Fe (III)) were prepared and employed in extracting of Phosvitin Phosphopeptides (short as PPPs) from egg yolk. It was found that the diameter of the magnetic CS nanoparticles was about 20 nm, and they could easily be aggregated by a magnet when suspending in the aqueous solution. The adsorption equilibrium of PPPs onto the obtained nanocarriers fitted well with the Langmuir model. The adsorption capacity of PPPs onto the superparamagnetic nanoparticles was influenced by pH and the initial concentration of the peptides solution. The final nitrogen/phosphorus molar ratios (short as N/P) of PPPs from crude egg yolk peptides and phosvitin peptides were low to 5.78 and 5.23, respectively. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and the higher purity of PPPs were obtained. In conclusion, this type of IMAN @ Fe (III) would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk.
Keywords: Chondroitin sulfate (CS); Phosvitin phosphopeptides (PPPs); Immobilized metal affinity nanoparticles (IMANs); Protein Purification; Metal ions;

A new HPLC UV validated method for therapeutic monitoring of deferasirox in thalassaemic patients by Silvia De Francia; Davide Massano; Francesca Maria Piccione; Elisa Pirro; Silvia Racca; Francesco Di Carlo; Antonio Piga (127-133).
We describe a new high performance liquid chromatography coupled with ultraviolet detection method for the quantification of plasma concentration of oral iron chelating agent deferasirox. A simple protein precipitation extraction procedure was applied on 500 μl of plasma aliquots. Chromatographic separation was achieved on a C18 reverse phase column and eluate was monitored at 295 nm, with 8 min of analytical run. This method has been validated following Food and Drug Administration procedures: mean intra and inter day variability was 4.64 and 10.55%; mean accuracy was 6.27%; mean extraction recovery 91.66%. Calibration curves ranged from 0.078125 to 40 μg/ml. Limit of quantification was set at 0.15625 while limit of detection at 0.078125 μg/ml. We applied methodology developed on plasma samples of thalassaemic patients treated with deferasirox, finding correlation between deferasirox plasma concentrations and serum ferritin levels. This methodology allowed a specific, sensitive and reliable determination of deferasirox, that could be useful to perform its therapeutic monitoring and pharmacokinetic studies in patients plasma.
Keywords: Deferasirox; HPLC UV; Quantification; Thalassaemic patients;

► A strategy for evaluating analyte adsorptive losses in urine samples is described. ► A range of additives, including phospholipids, were found to minimise losses. ► Elution of additives were monitored by LC–MS/MS. ► Impact on quantitative assay and metabolite identification was assessed.A key challenge in the development of robust bioanalytical methods, for the determination of drug analyte in human urine samples, is the elimination of potential analyte losses as a result of non-specific adsorption to container surfaces in which the samples are collected, stored or processed. A common approach to address adsorption issues is to treat the urine samples with additives that serve to increase analyte solubility and/or minimise interaction with the container surfaces. A series of adsorption experiments were performed on human urine samples containing an adsorption-prone in-house development compound (AZD9164). A roller-mixing methodology was employed to maximise sample interaction with container surfaces and quantification of analyte was performed by LC–MS/MS following minimal sample preparation. In the absence of any urine additive, adsorptive losses averaged 35% but were highly variable between different lots of urine. In the presence of a range of additives, including the surfactants Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and sodium dodecylbenzenesulphonate (SDBS), analyte adsorption was shown to be eliminated. Of particular academic interest was the finding that adsorptive losses could also be reduced upon the addition of phospholipid. The presence of additive generally had no marked impact on the analyte MS response but the use of an isotopically labelled internal standard satisfactorily compensated for instances in which ion suppression was observed, e.g. in the presence of Tween 80. Since metabolite profiling/identification investigations are often performed on urine samples originating from early clinical pharmacology studies, the elution of selected additives was also monitored by MS. CHAPS, dimethylacetamide (DMA) and HP-β-cyclodextrin eluted as single chromatographic peaks in, or just after, the column void volume whilst polymeric Tween 80, and to a lesser extent SDBS, eluted over a wide retention time window. The potential of the latter surfactants to obscure the detection of unknown metabolites is significant and therefore their use in urine samples, upon which metabolite investigations are to be performed, is not recommended. Upon consideration of other factors such as additive cost and toxicity, CHAPS was selected for use in development of the validated assay.
Keywords: LC–MS/MS; Analyte adsorption; Surfactant; Phospholipid; Bioanalysis; Metabolite identification;

Sensitive determination of isoprostanes in exhaled breath condensate samples with use of liquid chromatography–tandem mass spectrometry by Monika Janicka; Paweł Kubica; Agata Kot-Wasik; Jacek Kot; Jacek Namieśnik (144-149).
► Determination of isoprostanes as biomarkers in exhaled breath condensate samples. ► Analytical procedure for simultaneous separation of isoprostanes was developed. ► Higher levels of isoprostanes was determined in samples from tobacco smokers. ► Stability of isoprostanes was examined in different storage temperature.Oxidative stress is the hallmark of various inflammatory lung diseases. Increased concentrations of reactive oxygen species in the lungs are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. The aim of this work was to develop a method for the fast measurement of F2-isoprostanes in exhaled breath condensate (EBC) samples using equipment which is nowadays available and routinely exploited in analytical laboratories, liquid chromatography coupled with tandem mass spectrometry. Because of the limited volume of an EBC sample and the very low concentrations of biomarkers, we chose lyophilization as the preconcentration technique. The diastereoisomers determined show similar fragmentation patterns, which is why complete chromatographic separation with excellent peak shapes was essential for accurate quantitation. Isoprostanes were separated using a narrow-bore Agilent Extend C-18 column in isocratic elution mode using acetonitrile/methanol and water with the addition of 0.01%(v/v) formic acid. The limits of determination and quantitation for the determination of four isoprostanes in samples of EBC ranged from 1 to 3 pg/ml. The recoveries of all isoprostanes ranged from 96.7 to 101.7, with a relative standard deviation of <7%. The stability of the isoprostanes at different temperatures was measured as well.
Keywords: Exhaled breath condensates (EBC); Biomarkers; Isoprostanes; Liquid chromatography; Tandem mass spectrometry;

Validation of the PCR–dHPLC method for rapid identification of Candida glabrata phylogenetically related species in different biological matrices by O. Telleria; G. Ezpeleta; O. Herrero; I. Miranda-Zapico; G. Quindós; R. Cisterna (150-156).
► We tried to differentiate two new species (C. bracarensis and C. nivariensis) close related to C. glabrata with different phenotype and antifungal susceptibility profile. ► We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a fast and alternative novel technique for simultaneous identification of these three Candida species in different biological matrices. ► dHPLC is a suitable method for screening analysis to identify C. glabrata and its cryptic species.Since two new species phylogenetically related to Candida glabrata with slightly different phenotypes and antifungal susceptibility profiles have been described, it seems to be necessary from clinical point of view, to develop a rapid and accurate identification system in order to distinguish between these three fungal species. We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a faster (less than 7 min) and alternative novel technique for simultaneous analysis of Candida species in different biological matrices. The analyses show the good low limit of detection (LLOD) in all biological matrices studied (5.16–9.56 ng μL−1, 4.14–4.70 ng μL−1 and 3.99–4.66 ng μL−1 for Candida bracarensis, Candida nivariensis and C. glabrata, respectively). 180 Candida isolates were analyzed in order to demonstrate the method suitability for screening analysis to identify C. glabrata and its cryptic species (C. bracarensis and C. nivariensis) in clinical routine.
Keywords: Real-time PCR; dHPLC; C. glabrata; Cryptic species; Assay validation; Biological matrices;

A rapid and sensitive LC/ESI–MS/MS method for quantitative analysis of docetaxel in human plasma and its application to a pharmacokinetic study by Hiroaki Yamaguchi; Asuka Fujikawa; Hajime Ito; Nobuaki Tanaka; Ayako Furugen; Kazuaki Miyamori; Natsuko Takahashi; Jiro Ogura; Masaki Kobayashi; Takehiro Yamada; Nariyasu Mano; Ken Iseki (157-161).
► We developed a LC/ESI–MS/MS method for the determination of docetaxel in human plasma. ► One-step protein precipitation method was used for sample pretreatment without evaporation step. ► This method covered a linearity range of 5–5000 ng/mL with the lower limit of quantification of 5 ng/mL. ► The developed method was fully validated. ► This method was successfully applied for clinical pharmacokinetic investigation.Docetaxel is a taxane family antineoplastic agent widely employed in cancer chemotherapy. We developed a liquid chromatography/tandem mass spectrometry method for the determination of docetaxel in human plasma. Plasma samples were deproteinized by acetonitrile containing internal standard paclitaxel. Chromatographic separation was performed on a TSKgel ODS-100V 3 μm (50 mm × 2.0 mm i.d.) column using a mobile phase composed of acetonitrile–methanol–water–formic acid (50:5:45:0.1, v/v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. This method covered a linearity range of 5–5000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra-day precision and inter-day precision (R.S.D.) of analysis were less than 6.7%, and the accuracy (R.E.) was within ±9.0% at the concentrations of 5, 20, 200, and 2000 ng/mL. The total run time was 5.0 min. This method was successfully applied for clinical pharmacokinetic investigation.
Keywords: Docetaxel; LC/ESI–MS/MS; Rapid and sensitive analysis;

► Successfully applied to pharmacokinetic study in healthy volunteers. ► The method is simple, good specificity, robust and time efficient. ► The analytes of interest were well separated.A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of paracetamol (APAP) and its glucuronide conjugate (PG) in human plasma and urine. Plasma samples were precipitated with the mixture of acetonitrile and propylene glycol (90:10, v/v) solution and urine samples were diluted with the mobile phase, which were used to isolate the analytes from biological matrices followed by injection of the extracts onto a C18 column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI+). The method was validated over the concentration range of 10–30,000 ng/mL and 100–6000 ng/mL for APAP in human plasma and urine as well as 10–15,000 ng/mL and 200–60,000 ng/mL for PG in human plasma and urine, respectively. Inter- and intra-run precisions of APAP and PG were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 93.1% and 89.1% for APAP, and 93.7% and 92.3% for PG in human plasma and urine, respectively. The linearity, recovery and stability were validated for APAP and PG in human plasma and urine. The method proved to be simple, robust and time efficient.
Keywords: Paracetamol; Paracetamol glucuronide; Human plasma and urine; LC–MS/MS; Pharmacokinetic;

Development of a fast and simple liquid chromatography–tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples by Jeanne M. Rhea; Marion L. Snyder; Anne M. Winkler; Charbel Abou-Diwan; Corinne R. Fantz; James C. Ritchie; Fania Szlam; Kenichi A. Tanaka; Ross J. Molinaro (168-172).
► Direct UPLC–MS/MS method to quantitate argatroban in human plasma. ► UPLC–MS/MS offers a simple and quick alternative to indirect argatroban assay. ► Elevated fibrinogen affects indirect but not UPLC–MS/MS argatroban assay. ► Provides a wider analytical measurement range compared to currently published assays.An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC–MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100 μL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7 μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC–MS/MS method was linear over the concentration range of 0.003–3.0 μg/mL, with a lower limit of quantitation for argatroban of 0.003 μg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC–MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC–MS/MS method was unaffected. UPLC–MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.
Keywords: Mass spectrometry; Argatroban monitoring; Hemoclot Thrombin Inhibitors [HTI]; Plasma; Method comparison; Hospitalized patient samples; Heparin-Induced Thrombocytopenia (HIT);

Analysis of 8-hydroxy-2′-deoxyguanosine in human urine using hydrophilic interaction chromatography with tandem mass spectrometry by Chiemi Hosozumi; Akira Toriba; Thanyarat Chuesaard; Takayuki Kameda; Ning Tang; Kazuichi Hayakawa (173-176).
► Improved sensitivity of mass spectrometric detection by HILIC column. ► A sensitive and rapid analytical method for urinary 8-hydroxy-2′-deoxyguanosine. ► Strong chromatographic retention of polar compounds under highly organic mobile phase.Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used noninvasive biomarker of oxidative stress. A selective, sensitive and rapid method for determining 8-OHdG in human urine was developed using hydrophilic interaction chromatography–tandem mass spectrometry (HILIC–MS/MS) with electrospray ionization. 8-OHdG and isotopically labeled 8-OHdG (internal standard) were separated on a HILIC column with a mobile phase of 10 mM ammonium acetate: acetonitrile (1:9, v/v) within 10 min and detected by using a positive electrospray ionization interface under the selected reaction monitoring mode. The detection limits of 8-OHdG (corresponding to a signal-to-noise ratio of 3) for the HILIC-MS/MS system and the conventional method using a reversed-phase column with MS/MS were 1.0 and 26.0 fmol/injection, respectively. The proposed method makes it possible to monitor the basal level of urinary 8-OHdG from non-exposed healthy subjects and can be used for large-scale human studies.
Keywords: Hydrophilic interaction chromatography; Tandem mass spectrometry; 8-Hydroxy-2′-deoxyguanosine; Urine; Oxidative stress;

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS) by Yun-Gon Kim; Hyung-Yeon Park; Dongwon Yoo; Changmin Sung; Eunjung Song; Jae-Hun Lee; Yun-Hui Choi; Yong-Hyun Kim; Chang-Soo Lee; Kyungmoon Park; Byung-Gee Kim; Yung-Hun Yang (177-181).
► First application of derivatization reagents on the analysis of NDP-4K sugars with MS. ► Improvement of ESI analysis for NDP-4K sugar by derivatization. ► Finding of O-(2,3,4,5,6-pentafluoro benzyl) hydroxylamine as the best candidate. ► Possible future applications from the importance of NDP-4K sugars in the cell metabolism.Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.
Keywords: ESI-MS; Derivatization; Hydroxylamine; NDP-4-keto sugar; TDP-4-keto-6-deoxyglucose; NDP sugar;

► The successful PEGylation of CD133, a neural stem cell marker, is reported. ► The novel PEGylated CD133 can be used for the purification of neural stem cells. ► These findings are the first step in route to define a cell-purification bioprocess.Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I2–BaCl2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.
Keywords: CD133; Antibody PEGylation; Stem/progenitor cells separation; Streptavidin–biotin conjugation;

► A GC × GC method for assessing the fatty acid content of a cell culture line was developed. ► The method allowed identification of 15 new fatty acids present in insulin secreting cells, double the number previously known. ► Glucose effects on fatty acid content were determined and revealed decreases at high concentration for many fatty acids that may be related to secretion of fatty acids.A comprehensive two-dimensional gas chromatography (GC × GC) time-of-flight mass spectrometry method was developed for determination of fatty acids (irrespective of origin, i.e., both free fatty acids and fatty acids bound in sources such as triglycerides) in cultured mammalian cells. The method was applied to INS-1 cells, an insulin-secreting cell line commonly used as a model in diabetes studies. In the method, lipids were extracted and transformed to fatty acid methyl esters for analysis. GC × GC analysis revealed the presence of 30 identifiable fatty acids in the extract. This result doubles the number of fatty acids previously identified in these cells. The method yielded linear calibrations and an average relative standard deviation of 8.4% for replicate injections of samples and 12.4% for replicate analysis of different samples. The method was used to demonstrate changes in fatty acid content as a function of glucose concentration on the cells. These results demonstrate the utility of this method for analysis of fatty acids in mammalian cell cultures.
Keywords: Lipids; GC × GC; Insulin;

Influence of ionization source design on matrix effects during LC–ESI-MS/MS analysis by Chinmoy Ghosh; Chandrakant P. Shinde; Bhaswat S. Chakraborty (193-200).
► We design a model to study the matrix effects (ME) in different ion source design. ► Different phospholipids were identified in different ion sources causing ME. ► During the experiment Z-spray ion source coupled with UPLC showed more ME. ► Orthogonal spray ion source design coupled with HPLC showed less matrix effects. ► Scope of further research to study the role of ion source design on matrix effects.Glycerophosphocholines (GPChos) are known to cause matrix ionization effects during the analysis of biological samples (i.e. plasma, urine, etc.) in LC–MS/MS. In general, such matrix effect is directly related to an insufficient sample clean-up of the biofluids. In addition to GPCho; design of ionization source and/or LC also plays a very important role in matrix effects. In this research paper, different types of matrix effects, i.e. ion suppression or enhancement were observed in differently designed ion sources coupled with different LCs, from the same molecule, acamprosate (ACM), under the same chromatographic conditions. ACM was analyzed in a negative polarity in electrospray ionization interface using Z-spray and orthogonal spray ion source design. The analyte showed almost complete ion suppression in the Z-spray ionization source coupled with UPLC/HPLC, whereas there was very little ion enhancement in the orthogonal spray ionization source coupled with HPLC. In both the cases different GPChos were responsible, as evident from the presence of m/z 815.4 in Z-spray ion source and m/z 759.0 in orthogonal spray ion source. Hence, this approach can be used to evaluate the matrix effects in plasma samples during development and validation of LC–MS/MS method of drugs and their metabolites in different biological matrixes.
Keywords: LC–MS/MS; Matrix effect; Acamprosate; Phospholipids; Orthogonal spray; Z-Spray; Ion source design;