Journal of Chromatography B (v.912, #C)

Surface-modified anodic aluminum oxide membrane with hydroxyethyl celluloses as a matrix for bilirubin removal by Maoqiang Xue; Yisheng Ling; Guisen Wu; Xin Liu; Dongtao Ge; Wei Shi (1-7).
► An affinity membrane for bilirubin removal is fabricated using anodic aluminum oxide membrane as matrix. ► The affinity membranes have precise pore structure and narrow pore size distribution. ►The affinity membranes exhibit high adsorption capacity for bilirubin.Microporous anodic aluminum oxide (AAO) membranes were modified by 3-glycidoxypropyltrimethoxysilane to produce terminal epoxy groups. These were used to covalently link hydroxyethyl celluloses (HEC) to amplify reactive groups of AAO membrane. The hydroxyl groups of HEC-AAO composite membrane were further modified with 1,4-butanediol diglycidyl ether to link arginine as an affinity ligand. The contents of HEC and arginine of arginine-immobilized HEC-AAO membrane were 52.1 and 19.7 mg/g membrane, respectively. As biomedical adsorbents, the arginine-immobilized HEC-AAO membranes were tested for bilirubin removal. The non-specific bilirubin adsorption on the unmodified HEC-AAO composite membranes was 0.8 mg/g membrane. Higher bilirubin adsorption values, up to 52.6 mg/g membrane, were obtained with the arginine-immobilized HEC-AAO membranes. Elution of bilirubin showed desorption ratio was up to 85% using 0.3 M NaSCN solution as the desorption agent. Comparisons equilibrium and dynamic capacities showed that dynamic capacities were lower than the equilibrium capacities. In addition, the adsorption mechanism of bilirubin and the effects of temperature, initial concentration of bilirubin, albumin concentration and ionic strength on adsorption were also investigated.
Keywords: Affinity membrane; Hydroxyethyl celluloses; AAO membrane; Bilirubin;

► We developed a fast LC–MS method for polar metabolite analysis in plants. ► LC separation is achieved using a polar, reversed-phase column. ► Analyte detection is by orbitrap/ion trap mass spectrometry. ► All major sugars, amino acids and organic acids are quantified in a single run.Current liquid chromatography (LC) based methods for the analysis of polar plant metabolites require multiple runs using complex mobile phases and a combination of different columns. Here we describe a fast liquid chromatography–mass spectrometry (LC–MS) method for the determination of major polar metabolites in plants that requires only a single run using a single column. The method takes advantage of the ability to acquire both positive and negative data in an ion trap mass spectrometer (MS) and also the accurate mass capability of the orbitrap MS. The separation of polar compounds is achieved with a polar, reversed-phase column (Synergi Hydro-RP). A single analysis with a 25 min runtime is able to reliably determine the level of nearly all essential amino acids, several major organic acids and several major sugars in plant materials, as exemplified by analysis of a perennial ryegrass extract. The level of detection on column was below 0.1 ng (average 0.03 ng) for most amino acids, below 5 ng (average 2.3 ng) for organics acids and below 1 ng (average 0.64 ng) for sugars. The levels of quantified metabolites in ryegrass varied from 22 μg/g dry weight for histidine to 41 mg/g dry weight for sucrose.
Keywords: Polar compounds; LC–MS; Amino acids; Organic acids; Sugars; Plants;

► A sensitive method for analysis of raddeanin A in rat plasma is proposed. ► Method combined protein precipitation and LC–MS/MS. ► The pharmacokinetic study of raddeanin A in rats is firstly reported. ► The method offers a lower LOQ for raddeanin A than conventional methods.A simple and sensitive high-performance liquid chromatography–electro-spray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated to determine raddeanin A in rat plasma. After precipitation of rat plasma samples with methanol, chromatographic separation was achieved on a BDS Hypersil C18 column (100 × 2.1 mm, 2.4 μm) using the mobile phase consisted of acetonitrile and 2 mM ammonium acetate with 0.05% formic acid (60:40, v/v). The detection was performed in a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode using negative ionization. The transition monitored were m/z 895.6 → 455.0 for raddeanin A and m/z 359.3 → 329.0 for IS, respectively. The method was linear over the concentration range of 2–1000 ng/mL for raddeanin A. The intra-day and inter-day assay variations were <9.46%, and the accuracy values were between −2.04% and −6.52% relative error. The extraction recovery of raddeanin A was more than 70%, and the relative matrix effect ranges from 108.52% to 112.36%. The validated method has been successfully applied to determine the pharmacokinetic profile of raddeanin A in rat plasma following oral and intravenous administration.
Keywords: Raddeanin A; LC–MS/MS; Pharmacokinetics; Absolute bioavailability;

► A validated method describing the extraction of plasma enterolignans with supported liquid extraction (SLE). ► SLE as a preferred extraction technique to LLE for enterolignan isolation. ► Dietary flaxseed significantly raised plasma enterolignan levels after 4 weeks.Dietary interventions involving foods that are enriched in lignans, such as flaxseed, are drawing attention due to their beneficial protective effects in various diseases and human conditions. Accurate quantitation of key lignan metabolites such as enterodiol (END) and enterolactone (ENL) is necessary in order to identify factors that may influence overall bioavailability. Here we describe the validation of a novel supported liquid extraction (SLE) method for isolation of plasma enterolignans, END and ENL, using 2H6-labeled isotopes with gas chromatography–mass spectrometry in micro selected ion storage (GC/MS-μSIS) mode. Following enzymatic hydrolysis and SLE extraction with 70:30 diethyl ether:ethyl acetate, enterolignans were rapidly separated within 8 min. SLE in combination with GC/MS-μSIS gave high recoveries of 96.4% and 96.0% for END and ENL. Intra-assay precision ranged from 2.5 to 5.9% for both compounds whereas the inter-assay precision was 2.6–6.9%. SLE was also directly compared to liquid liquid extraction (LLE). Both techniques offered high precision and accuracy, however, SLE consistently enabled successful analyte extractions and derivatizations, unlike LLE, which had an ∼4% failure rate. SLE was also tested in a study where dietary milled flaxseed supplementation (30 g/day for 1 month) and enterolignan bioavailability was examined in a healthy, human population (n  = 10). Plasma total enterolignan levels significantly increased (P  = 0.002) at 4 weeks relative to baseline. Average concentrations for END and ENL were 209 nM and 304 nM, respectively.
Keywords: Enterolignans; Plasma; Supported liquid extraction; Gas chromatography; Mass spectrometry; Flaxseed;

► Magnetic solid-phase extraction was developed for determination of puerarin in rat plasma. ► Puerarin adsorbed with Fe3O4@SiO2-C18 sorbents could be simply isolated. ► Method validations were studied. ► The novel extraction method is useful for pharmaceutical analysis.In the paper, we presented a magnetic solid-phase extraction (MSPE) method based on C18-functionalized magnetic silica nanoparticles for the analysis of puerarin in rat plasma. The approach involves two steps including synthesis of magnetic solid-phase sorbents and bioanalysis. The synthesized magnetic silica microspheres modified with chloro(dimethyl)octylsilane (namely Fe3O4@SiO2-C18) can provide an efficient way for the extraction of puerarin through C18 hydrophobic interaction. The puerarin could be easily enriched using milligram-level Fe3O4@SiO2-C18 sorbents with vibration for 10 min. By means of a magnet, puerarin adsorbed with Fe3O4@SiO2-C18 sorbents was easily isolated from the matrix, and desorbed with CAN. No carryover was observed, and the sorbents could be recycled in our study. The method recoveries were obtained from 85.2% to 92.3%. Limits of quantification and limits of detection of 0.1 μg mL−1 and 0.05 μg mL−1, respectively were achieved. The precision was from 8.1 to 13.7% for intra-day measurement, and from 9.4 to 15.2% for inter-day variation. The accuracy ranged from 94.7 to 106.3% for intra-day measurement, and from 93.3 to 107.8% for inter-day measurement. The MSPE method was applied for analysis of puerarin in rat plasma samples. The results indicated that it was convenient and efficient for the determination of puerarin in biosamples.
Keywords: Magnetic solid-phase extraction; Puerarin; Rat plasma; High performance liquid chromatography;

► LF-3-88, a novel agonist of α4β2*-nAChRs, is being developed as an antidepressant. ► A quantitative method based on UHPLC–MS–MS was developed and validated for LF-3-88. ► LF-3-88 was measured in mouse plasma and brain tissue after oral administration. ► Pharmacokinetics of LF-3-88 indicated good bioavailability and brain penetration.LF-3-88 (2-[5-[5-(2(S)-azetidinylmethoxyl)-3-pyridyl]-3-isoxazolyl]ethanol) was identified as a highly selective α4β2-nAChRs partial agonist, with a K i value of 0.4 nM and EC50 value of 110 nM. A sensitive and selective ultra high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) method was developed and validated to study the pharmacokinetics profile of this compound in mice. Protein precipitation with acetonitrile was used to prepare the plasma and brain samples, and the recovery was greater than 90%. The inter-day and intra-day accuracy and precision of the quantitative method ranged from 95% to 106% for plasma and from 93% to 105% for brain homogenates. The precision of the assay was <10%. The limit of detection and limit of quantitation were 0.5 ng/mL (1.8 nM) and 1 ng/mL (3.6 nM), respectively. LF-3-88 was stable (>93%) for 24 h on the bench top at room temperature, and for at least 3 weeks at 4 °C and −80 °C. The UHPLC–MS–MS assay was applied to the measurement of plasma and brain levels of LF-3-88 following oral administration to male Balb/c mice. Plasma concentrations of LF-3-88 and brain levels were dose-dependent with half-lives of approximately 60 min and 180 min, respectively, indicating good oral bioavailability and penetration of the blood–brain barrier.
Keywords: α4β2-nAChRs partial agonist; Antidepressant; UHPLC–MS–MS; Pharmacokinetics; Quantitative analysis;

LC–MS/MS method for determination of geldanamycin derivative GM-AMPL in rat plasma to support preclinical development by Yan-ping Li; Lin-yan Gao; Kai-tong Li; Shuai Meng; Jian-hua Zhu; Dong Li; Jie Jin; Guang-zhi Shan; Zhuo-rong Li (43-49).
► First report on oral bioavailability of novel Hsp90 inhibitor GM-AMPL. ► The sensitive and reliable LC–MS/MS method for determination of GM-AMPL in rat plasma. ► Simple and stable extraction procedure of analytes from plasma samples. ► Validated method feasible to future pharmacokinetic study.A LC–MS/MS method for determining the concentration of the small molecule Hsp90 inhibitor, GM-AMPL, has been developed and validated in rat plasma to support preclinical development. 17-[2-(morpholine-4-yl)ethyl]amino-17-demethoxygeldanamycin (GM-AMPL) and the internal standard 17-allylamino-17-demethoxygeldanamycin (17-AAG) were sufficiently separated on a Venusil MP C18 column that was eluted with 80% methanol in water at 40 °C. Quantification studies were performed with a multiple reaction monitoring (MRM) transition of m/z 657.3→614.3 and 584.3→541.3 for GM-AMPL and IS, respectively, in the negative ion mode. The present method exhibited good linearity (R  > 0.999) over the concentration range of 2–600 ng/mL for GM-AMPL in rat plasma with a lower limit of quantification (LLOQ) of 2 ng/mL. The intra-batch and inter-batch assay coefficients of variation (CV) were in range of 1.56–6.84% and 1.62–6.98%, respectively. The plasma samples were extracted with methanol to precipitate protein with extraction recovery in range of 84.09–95.25%. The matrix effect was determined as internal substance (IS) normalized matrix factor of 1.09, 1.18 and 1.05 for samples with three concentration levels of 4, 40 and 400 ng/mL, respectively. This validated method was further applied to successfully determine the pharmacokinetic parameters and oral availability of GM-AMPL in Sprague-Dawley rats following intravenous injection and oral administration.
Keywords: LC–MS/MS; Geldanamycin derivative; Pharmacokinetics; Plasma concentration;

► BMC methodology as an early predictor of protein–drug binding. ► Ability of SDS to form a bio-mimetic protein similar to α1-acid glycoprotein. ► Usefulness of SDS in BMC for predicting protein binding of basic and neutral drugs.A simple and fast method is of urgent need to measure protein–drug binding affinity in order to meet the rapid development of new drugs. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography (MLC) using micellar mobile phases in adequate experimental conditions, can be useful as an in vitro system in mimicking the drug–protein interactions. In this study, sodium dodecyl sulfate–micellar liquid chromatography (SDS–MLC) was used for the prediction of protein–drug binding based on the similar property of SDS micelles to α1-acid glycoprotein (AGP). The relationships between the BMC retention data of a heterogeneous set of 14 basic and neutral drugs and their plasma protein binding parameter were studied and the predictive ability of models was evaluated. Modeling of log  k BMC of these compounds was established by multiple linear regression (MLR) and second-order polynomial models obtained in two different concentrations (0.07 and 0.09 M) of SDS. The developed MLR models were characterized by both the descriptive and predictive ability (R 2  = 0.882, R CV 2 = 0.832 and R 2  = 0.840, R CV 2 = 0.765 for 0.07 and 0.09 M SDS, respectively). The p values <0.01 also indicated that the relationships between the protein–drug binding and the log  k BMC values were statistically significant at the 99% confidence level. The standard error of estimation showed the standard deviation of the regression to be 11.89 and 13.87 for 0.07 and 0.09 M, respectively. The application of the developed model to a prediction set demonstrated that the model was also reliable with good predictive accuracy. The external and internal validation results showed that the predicted values were in good agreement with the experimental value.
Keywords: Protein–drug binding; α1-Acid glycoprotein (AGP); Biopartitioning micellar chromatography; Sodium dodecyl sulfate (SDS);

► Carryover effects is an underestimated phenomenon in proteomic research. ► Analytical column represents the main source of carryover in the HPLC system. ► Carryover effects can still be observed after executing five gradient blanks. ► Capillary column packed with porous particles showed higher effects than monoliths.This article relates on reversed-phase column technology as the main cause of carryover in the LC–MS/MS analysis of proteomics samples. The separation performance and column carryover was investigated using four capillary columns with different morphologies by monitoring the remaining traces of tryptic peptides of bovine serum albumin in subsequent blank LC–MS runs. The following trend in column carryover was observed: capillary column packed with 3 μm porous C18 particles ≫ 2.7 μm fused-core C18 packed column > silica C18 monolith ≫ poly(styrene-co-divinylbenzene) monolith. This is mainly related to the intrinsic properties of the different chromatographic materials, related to surface area and the presence and size of mesopores (stagnant zones where mass transfer is controlled by diffusion). Both isocratic and gradient wash steps with 2-propanol/acetonitrile mixtures were not effective to reduce column carryover. An isocratic wash step using a high acetonitrile percentage or blank gradient reduced carryover with approximately 50%. Nevertheless, it is important to note that effects of column carryover were still observed in a fifth subsequent gradient blank. Although the polymer monolith clearly outperformed the silica materials in terms of carryover, this material exhibited also the lowest loadability, which may be a disadvantage when profiling proteomics mixtures with a broad dynamic range.
Keywords: Column technology; Proteomics; LC–MS; Column comparison;

► We have adopted surface initiated electropolymerization of benzidine monomer. ► This approach takes advantage of the nanostructured ultrathin imprinted film. ► Successful enantiodifferentiation and ultratrace analysis of d- and l-methionine. ► Stringent detection limit without any non-specific false-positive contribution.An alternative method is presented for the modification of pencil graphite fibers using surface imprinting technology. In this new approach, we have adopted surface initiated electropolymerization of benzidine monomer, with simultaneous imprinting of template (d- and l-methionine), on carboxylated multiwalled carbon nanotubes anchored pencil graphite fiber. This yielded a nanostructured ultrathin imprinted film (58.3 nm) uniformly coated all along the perimeter and length of pencil graphite fiber, for micro-solid phase extraction with substantial adsorption capability. The same film is coated over the exposed tip of the pencil graphite fiber to serve as a complementary molecularly imprinted polymer-sensor. Both extraction and sensing devices are not capable to measure the stringent limit (0.016 ng mL−1) of clinical detection of methylenetetrahydrofolate reductase (MTHFR) gene mutation caused by acute methionine depletion, when used alone. However, on combination of both techniques, a successful enantioselective analysis of d- and l-methionine with excellent analytical figures of merit [limit of quantitation range: 0.03–30.00 ng mL−1, limit of detection: 0.0098 ng mL−1 (RSD = 2.04, S/N  = 3)] could be achieved without any problem of non-specific false-positive contribution and cross-reactivity, in real samples.
Keywords: Electropolymerization; d- and l-methionine; Molecularly imprinted micro-solid phase extraction pencil graphite fiber; Enantioseparation; Differential pulse cathodic stripping voltammetry;

► LC–ESI-MS/MS utilized for PK study of six hydrophilic components in Jitai tablet. ► LC–ESI-MS/MS accomplished high–resolution separation and provided lower LLOQs. ► The PK properties of the six components were characterized as rapid oral absorption. ► The method is simple, fast, specific and sensitive.A liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method was developed and validated for the simultaneous determination of amygdalin (ADL), danshensu (DSS), ferulic acid (FA), hydroxysafflor yellow A (HSYA), salvianolic acid A (SAA) and salvianolic acid B (SAB) in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. LC separation was performed on a Zorbax Eclipse Plus C18 column (3.0 mm × 100 mm I.D, 1.8 μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3 mL/min. ESI-MS spectra was acquired in negative ion multiple reaction monitoring mode. The mass transition ion-pair was followed as m/z 456.0 → 323.1, m/z 197.3 → 178.8, m/z 193.0 → 133.9, m/z 611.1 → 325.2, m/z 493.0 → 295.0, and m/z 717.0 → 519.0 for ADL, DSS, FA, HSYA, SAA and SAB, respectively. All analytes showed good linearity over a wide concentration range (r  > 0.99). The lower limit of quantification was 7 ng/mL, 2 ng/mL, 4 ng/mL, 1 ng/mL, 2 ng/mL, and 4 ng/mL for ADL, DSS, FA, HSYA, SAA and SAB, respectively. The mean recovery of the analytes ranged from 86.29% to 93.16%. The intra- and inter-day precisions were in the range of 1.50–9.98% and the accuracies were between 91.17% and 99.46%. The validated method was successfully applied to a pharmacokinetic study of the six hydrophilic components in rat plasma after oral administration of Jitai tablet.
Keywords: Hydrophilic component; Jitai tablet; LC–ESI-MS/MS; Pharmacokinetic study;

► We built a FGFR4/CMC-online-HPLC/MS system to screening active components from traditional Chinese medicine. ► Sinapine was identified as active compounds in total extracts of Brasscia albla L. ► The binding sites of sinapine and gefitinib was the tyrosine kinase domain of FGFR4 based on competitive displacement assay and FleX dock simulation. ► sinapine could inhibit cell proliferation with dose-dependent.We investigated an analytical method for the recognition separation, and identification of active components from the traditional Chinese medicinal plant Brassica albla L. using fibroblast growth factor receptor 4 cell membrane chromatography (FGFR4/CMC) with high performance liquid chromatography/mass spectrometry (HPLC/MS). HEK293-FGFR4 cells were obtained by stable transfection of the HEK293 cell line with pcDNA3.1 vector containing the FGFR4 gene. Crude extracts of B. albla L. were firstly subjected to FGFR4/CMC column, and the retain contents on the FGFR4/CMC column were then transferred and enriched using a pre-column, and a ten port column switcher were used between FGFR4/CMC column and HPLC. The retained components on FGFR4/CMC column were then directly delivered to the HPLC/MS system for separation and identification. Gefitinib, nicotine, atenolol, and nimodipine were used in order to verify FGFR4/CMC-HPLC/MS system specificity. Subsequently, we investigated the reproducibility and reliability of the FGFR4/CMC-HPLC/MS system. Finally, sinapine was identified as an active component of B. albla L. The MTT colorimetric assay revealed sinapine could inhibit the proliferation of HEK293-FGFR4 cells with dose dependence. Competitive displacement assay approved getitinib could occupy binding site of sinapine with competition way. And FleX dock simulation further exhibited sinapine and gefitinib could bind with the FGFR4 tyrosine active domain. Thus, sinapine is a potential tumor antagonist that acts on the tyrosine kinase domain.
Keywords: Fibroblast growth factor receptor 4; Cell membrane chromatography; High performance liquid chromatography/mass spectrometry; Brassica albla L.; Sinapine;

Ultra-sensitive assay for paclitaxel in intracellular compartments of A549 cells using liquid chromatography–tandem mass spectrometry by Tingting Wang; Wenxiao Ma; Yantong Sun; Yan Yang; Weiping Zhang; J. Paul Fawcett; Hongwei Du; Jingkai Gu (93-97).
► The LLOQ of this assay is 2 pg/mL, which is lower than ever before (20 pg/mL). ► The common method to determine the anticancer drugs binding to microtubules is radioactive analysis. ► Radioactive analysis could affect the normal physiological state of cells and could not differentiate drug and metabolites. ► This method is simple and reproducible, diminit matrix interferences.A high-performance liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of paclitaxel in intracellular compartments using docetaxel as internal standard (IS) has been developed and validated. A549 cancer cells (106) were incubated with paclitaxel (2 ng/mL) for up to 4 h and then subjected to sequential extraction of cytosolic, membrane/organelle, nuclear and cytoskeleton soluble protein. Fractions were ultrasonicated to release protein bound paclitaxel after which drug was extracted using liquid–liquid extraction with diethyl ether:dichloromethane (2:1, v/v). Chromatographic separation was then carried out on an Ascentis Express C18 column (50 mm × 4.6 mm, 2.7 μm) with a mobile phase of acetonitrile:0.1% formic acid in water (50:50, v/v). Detection involved electrospray positive ionization followed by multiple reactions monitoring of the precursor-to-product ion transitions of paclitaxel at m/z 854.4 → 286.3 and docetaxel at m/z 808.6 → 226.1. Assay validation based on samples of total cell extract in the same buffer as protein fractions showed the assay was linear over the range 2–600 pg/mL with intra- and inter-day precision (as relative standard deviation) and accuracy (as relative error) of <7% and <±12%, respectively. Recovery was approximately 70% and matrix effects were minimal. The distribution of paclitaxel in subcellular components of A549 cancer cells was mainly into the cytoskeletal compartment.
Keywords: Paclitaxel; Liquid–liquid extraction; LC–MS/MS; A549 cells; Intracellular distribution;

► Electrospun crosslinked PVA as UTLC stationary phase. ► Eleven laser dye labeled amino acids were tested on the PVA UTLC plate. ► The separation efficiency on PVA UTLC plate was much higher than on Si-Gel HPTLC plate. ► Unlabeled amino acids, alanine, methionine, arginine, and phenylalanine were baseline separated on PVA UTLC plate using ninhydrin as visualization reagent.Electrospun polyvinyl alcohol (PVA) ultrathin layer chromatographic (UTLC) plates were fabricated using in situ crosslinking electrospinning technique. The value of these ULTC plates were characterized using the separation of fluorescein isothiocyanate (FITC) labeled amino acids and the separation of amino acids followed visualization using ninhydrin. The in situ crosslinked electrospun PVA plates showed enhanced stability in water and were stable when used for the UTLC study. The selectivity of FITC labeled amino acids on PVA plate was compared with that on commercial Si-Gel plate. The efficiency of the separation varied with analyte concentration, size of capillary analyte applicator, analyte volume, and mat thickness. The concentration of 7 mM or less, 50 μm i.d. capillary applicator, minimum volume of analyte solution and three-layered mat provides the best efficiency of FITC-labeled amino acids on PVA UTLC plate. The efficiency on PVA plate was greatly improved compared to the efficiency on Si-Gel HPTLC plate. The hydrolysis products of aspartame in diet coke, aspartic acid and phenylalanine, were also successfully analyzed using PVA-UTLC plate.
Keywords: Planar chromatography; Amino acids; Nanofiber; Electrospin;

► We developed a comprehensive analytical method based on HPLC–MS to analyze mitochondrial phospholipids. ► We optimized extraction and simplification procedures. ► We developed normal phase HPLC conditions for a rigorous quantitation with an internal standard and standard curves. ► We detected and quantitated low abundance phospholipids such as monolysocardiolipin and phosphatidylglycerol.A normal-phase HPLC–MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.
Keywords: Normal-phase HPLC; Mass spectrometry; Solid phase extraction; Internal standard; Standard curve; Ion suppression; Cardiolipin; Monolysocardiolipin;