Capillary-Channeled Polymer (C-CP) Films as Processing Platforms for Protein Analysis by Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry (MALDI-MS)

Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, μm-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 μL of 2.5 μM each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H₂O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.     

分子印迹空心球在蛋白质固相萃取中的应用

建立了分子印迹固相萃取-高效液相色谱相结合分离复杂样品中牛血红蛋白(Hb)的方法. 以单分散的二氧化硅球为载体, Hb为模板, 采用表面印迹法制备了分子印迹空心球(MIHS). 采用透射电子显微镜(TEM)对该印迹材料的形貌进行了表征. 结果表明, 所制备的MIHS的单分散性好、 粒径均匀, 是一种理想的固相萃取填料. 以该印迹材料作为固相萃取吸附剂, 并结合高效液相色谱(HPLC)研究了其对Hb的吸附行为, 结果表明, 制备的MIHS在磷酸盐缓冲液(pH=7.0)中对Hb的回收率高于80%, 而对核糖核酸酶A(RNase A)和细胞色素C(Cyt C)的回收率低于50%, 方法的精密度高(RSD<5%). 与Hb印迹硅球相比, MIHS具有较快的传质速率和萃取效率. 将MHIS应用于复杂样品中Hb的分离, 结果表明, MIHS可以从加标牛血清中选择性吸附Hb, 吸附率达82%.     

Microbore polypropylene capillary channeled polymer (C-CP) fiber columns for rapid reversed-phase HPLC of proteins

The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s(-1) at 1.5 mL min(-1)). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5%), but increased resolution of ~14% between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s(-1) required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.     

Determination of asymmetric and symmetric dimethylarginines in human plasma by HPLC with electrochemical detection

A new HPLC method was developed and validated for the determination of asymmetric and symmetric dimethylarginines and l ‐arginine in human plasma. After SPE and evaporation of the eluate, the samples were derivatised with an o‐phthaldialdehyde reagent containing 3‐mercaptopropionic acid. The derivatives formed were analysed by isocratic RP‐HPLC with electrochemical detection at +320 mV. The mobile phase consisted of 50 mM phosphate buffer (pH 6.1) containing 10% v/v acetonitrile, the flow rate was 1 mL/min. The retention times of all compounds including monomethylarginine (internal standard) were <24 min. The LODs (S/N 3:1) were 0.012 μM for both dimethylarginines and 0.013 μM for l ‐arginine; the linearity of the method was from 0.1 to 20 μM for both dimethylarginines and from 1 to 200 μM for l ‐arginine. Absolute extraction recoveries measured for all analytes ranged from 85 to 88%.     

Piroxicam quantitation in human plasma by high-performance liquid chromatography with on- and off-line solid-phase extraction.

A comparative study of two analytical methodologies for piroxicam quantitation in plasma by off-line and on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contained C8 for both extraction methods. The analytes piroxicam and tenoxican (internal standard) were separated on a C18 column with a mobile phase consisting of acetonitrile:20 mM phosphate buffer pH 3.1 (50:50, v/v) followed by UV detection at 360 nm. The validation of the methods demonstrated good recoveries (over 90%), sensitivity (limits of quantification of 0.05 microgram/ml with on-line SPE and 0.1 microgram/ml with off-line SPE, based on a 100 microliters and 200 microliters sample volume, respectively), accuracy and precision (better than 9.5%). Both methodologies have been used for bioequivalence studies.     

Capillary electrophoresis–electrochemiluminescence detection method for the analysis of ibandronate in drug formulations and human urine

A simple, rapid and sensitive CE method coupled with electrochemiluminescence (ECL) detection for direct analysis of ibandronate (IBAN) has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 9.0) and a voltage of 13.5 kV, separation of IBAN in a 30‐cm length capillary was achieved in 3 min. ECL detection was performed with an indium tin oxide working electrode bias at 1.6 V (versus a Pt wire reference) in a 200‐mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)₃²⁺ (where bpy=2,2′‐bipyridyl). Derivatization of IBAN prior to CE‐ECL analysis was not needed. Linear correlation (r=0.9992, n=7) between ECL intensity and analyte concentration was obtained in the range of 0.25–50 μM IBAN. The LOD of IBAN in water was 0.08 μM. The developed method was applied to the analysis of IBAN in a drug formulation and human urine sample. SPE using magnetic Fe₃O₄@Al₂O₃ nanoparticles as the extraction phase was employed to pretreat the urine sample before CE‐ECL analysis. The linear range was 0.2–12.0 μM IBAN in human urine (r=0.9974, n=6). The LOD of IBAN in urine was 0.06 μM. Total analysis time including sample preparation was <1 h.     

Application of polydopamine-coated nylon capillary-channeled polymer fibers as a stationary phase for mass spectrometric phosphopeptide analysis

Capillary-channeled polymer (C-CP) fibers are demonstrated as a selective stationary phase for phosphopeptide analysis via LC–MS. Taking advantage of the oxidative self-polymerization of dopamine under alkaline conditions, a simple system involving a dilute aqueous solution of 0.2% w/v dopamine hydrochloride in 0.15% w/v TRIS buffer, pH 8.5 was utilized to coat polydopamine onto nylon 6 C-CP fibers. Confirmation of the polydopamine coating on the fibers (nylon-PDA) was made through attenuated total reflection-FTIR (ATR-FTIR) analysis. Imaging using SEM was also performed to examine the morphology and topography of the nylon-PDA. Subsequent loading of Fe³⁺ to the nylon-PDA matrix was confirmed by SEM/energy dispersive X-ray spectroscopy (SEM/EDX). The Fe³⁺-bound nylon-PDA fibers packed in a microbore column format were tested in the off-line preconcentration of phosphopeptides from a 1:100 mixture of β-casein/BSA digests for MALDI-TOF analysis. The packed column was also installed onto an HPLC system as a platform for the online sample clean-up and enrichment of phosphopeptides from a 1:1000 mixture of β-casein/BSA protein digests that were determined by subsequent ESI–MS analysis.     

Multifunctional protein processing chip with integrated digestion, solid-phase extraction, separation and electrospray

We describe a microfluidic device in which integrated tryptic digestion, SPE, CE separation and electrospray ionization for MS are performed. The chip comprised of 10 × 30 μm channels for CE, and two serially connected 150?μm deep, 800?μm wide channels packed with 40 to 60 μm diameter beads, loaded with either immobilized trypsin, reversed-phase packing or both. On-chip digestion of cytochrome c using the trypsin bed showed complete consumption of the protein in 3 min, in contrast to the 2 h required for conventional solution phase tryptic digestion. SPE of 0.25 μg/mL solutions of the peptides leu-enkephalin, angiotensin II and LHRH gave concentration enhancements in the range of 4.4-12, for a ten times nominal volume ratio. A 100 nM cytochrome c sample concentrated 13.3 times on-chip gave a sequence coverage of 85.6%, with recovery values ranging from 41.2 to 106%. The same sample run without SPE showed only five fragment peaks and a sequence coverage of 41.3%. When both on-chip digestion and SPE (13.3 volume ratio concentration enhancement) were performed on 200 nM cytochrome c samples, a sequence coverage of 76.0% and recovery values of 21-105% were observed. Performing on-chip digestion alone on the same sample gave only one significant fragment peak. The above digestion/peptide concentration step was compared to on-chip protein concentration by SPE followed by on-chip digestion with solution phase trypsin. Both procedures gave similar recovery results; however, much larger trypsin autodigestion interference in the latter approach was apparent.     

Determination of ng/mL levetiracetam using ultra-high-performance liquid chromatography-photodiode absorbance

This paper demonstrates the analysis of levetiracetam, a new chiral antiepileptic drug, at ng/mL levels using an ultra-high-performance liquid chromatography (UHPLC)-photodiode absorbance (PDA) method. Three different sample preparation methods, liquid-liquid extraction with Extrelut, solid phase extraction (SPE) with Oasis HLB and Oasis MAX SPE cartridges, and protein precipitation with organic solvents were carried out. The last preparatory method is the simplest and provides the best recoveries: between 97.1% and 100.4% with RSD value below 5%. The column for separation is BEH C18 column (1.7 μm particle size and 100 × 2.1 mm i.d.) and acetonitrile-phosphate buffer (pH = 6.6; 0.01 M) (10/90 v/v) is the mobile phase. The results obtained are compared to analysis conducted by the HPLC method. The UHPLC method was validated in the range of 2-100 μg/mL levetiracetam concentration (R(2) = 0.9997). LOD and LOQ are 10 ng/mL and 33 ng/mL, respectively. The developed UHPLC method was applied to plasma samples of patient with epilepsy.     

Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000-12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics.     

A novel stationary phase: capillary-channeled polymer (C-CP) fibers for HPLC separations of proteins

A novel stationary phase is demonstrated for the separation of proteins. Capillary-channeled polymer (C-CP) fibers provide a stationary phase that is characterized by a high surface activity (yielding strong wicking action) and drastically reduced back pressures. Columns prepared by pulling approximately 1200 50- micro m diameter polypropylene C-CP fibers through stainless steel tubing with column dimensions of 4.6-mm i.d. and 306-mm length exhibit reversed-phase characteristics in the separation of the proteins. A gradient method [95:5 water-acetonitrile (ACN)/propanol (1:1) to 35:65 water-ACN/propanol] with trifluoroacetic acid added as an ion-pairing agent yields high-quality separations of superoxide dismutase, hemoglobin, hemocyanin, and myoglobin. It is believed that the C-CP fiber stationary phase holds a number of promising traits for applications in both analytical and prep-scale separations of diverse organic species, including a wide range of biomolecules.     

In-Line Desalting of Proteins from Buffer and Synthetic Urine Solution Prior to ESI-MS Analysis via a Capillary-Channeled Polymer Fiber Microcolumn

Presented here is a novel in-line solid phase extraction (SPE) method utilizing a capillary-channeled polymer (C-CP) fiber microcolumn prior to introduction to an electrospray ionization (ESI) source. The high permeability of the microcolumn allows for operation under syringe pump or HPLC driven flow, ultimately providing greater mass spectral clarity and accurate molecular weight determinations for different protein/buffer combinations. Studies presented here focus on the desalting of several target proteins from a standard phosphate buffered saline (PBS) matrix and a synthetic urine solution prior to ESI-MS determinations. In every case, responses for μM-level proteins in PBS improve from the situation of not permitting molecular weight determinations to values that are precise to better than ±10 Da, without internal standards, with relative improvements in the signal-to-background ratios (S/B) on the order of 3,000×. De-salting of a myoglobin-spiked (12 μM) synthetic urine results in equally-improved spectral quality.      

A capillary zone electrophoresis for determination of thiolic peptides in biological samples

A new method to improve the analyses of thiolic peptides (cysteine, γGlu-Cys, glutathione, phytochelatins and desglycyl-phytochelatins) derivatized with monobromobimane (mBrB) in complex biological samples by CZE is described. The method involves a SPE using Sep-Pak Light C18 Cartridges after derivatization and a later CZE analysis. Elution of mBrB-thiols was achieved with 10 mM HCl + 70% methanol v/v in deionised water. Electrophoretic parameters, such as BGE pH and concentration, different organic additives (methanol and trifluoroethanol), applied voltage and capillary length were studied in order to establish suitable analytical conditions. Optimum separation of the mBrB-thiolic peptides was obtained with 100 mM sodium borate buffer at pH 7.60. The electrophoretic conditions were +15 kV, capillary length of 90 cm from inlet to detector (98 cm total length, 50 μm ID), samples were loaded into the capillary by hydrodynamic injection (50 mbar, 20 s) and detection was performed at 390 nm. The improved method showed good reproducibility, linearity and sensitivity. The LODs and LOQs estimated using a standard of GSH were 1.41 and 4.69 μM respectively.     

Determination of pesticides in wine using micellar electrokinetic chromatography with UV detection and sample stacking

In this work, the analysis of a group of four fungicides (pyrimethanil, nuarimol, procymidone and cyprodinil) and one insecticide (pirimicarb) by micellar electrokinetic chromatography (MEKC) with UV detection using the on-line preconcentration strategy called reversed electrode polarity stacking mode (REPSM) is proposed. After optimisation, an adequate separation electrolyte for the separation and stacking of these pesticides was obtained which consisted of 100 mM borate, 60 mM sodium dodecyl sulphate (SDS), at pH 9.0 and 2% 2-propanol. The use of this running buffer together with the REPSM preconcentration method provided limits of detection (LODs) between 38.3 and 241 microg/L. In order to apply the developed methodology for the analysis of these pesticides in wine samples, several off-line preconcentration strategies (mainly, solid-phase extraction, SPE, and solid-phase microextraction, SPME) were tested. Although the use of a SPE procedure, optimized in this work for water samples, using Oasis HLB cartridges, provided mean recovery values between 79 and 100% for spiked water samples, it could not be applied to the extraction of these pesticides from wine samples due to high interference from the sample matrix. However, the use of a SPME procedure using polydimethylsiloxane/divynilbenzene (PDMS/DVB) fibers allowed the selective extraction of four of the five pesticides which could be perfectly determined. The final combination of the off-line SPME and on-line REPSM preconcentration strategies allowed obtaining LODs between 17.6 and 32.3 microg/L.     

Mechanisms of NO release by N1-nitrosomelatonin: nucleophilic attack versus reducing pathways

A new type of physiologically relevant nitrosamines have been recently recognized, the N(1)-nitrosoindoles. The possible pathways by which N(1)-nitrosomelatonin (NOMel) can react in physiological environments have been studied. Our results show that NOMel slowly decomposes spontaneously in aqueous solution, generating melatonin as the main organic product (k = (3.7 +/- 1.1) x 10(-5) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C, anaerobic). This rate is accelerated by acidification (k(pH 5.8) = (4.5 +/- 0.7) x 10(-4) s(-1), k(pH 8.8) = (3.9 +/- 0.6) x 10(-6) s(-1), Tris-HCl (0.2 M) buffer at 37 degrees C), by the presence of O(2) (k(o) = (9.8 +/- 0.1) x 10(-5) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, P(O(2)) = 1 atm), and by the presence of the spin trap TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl; k(o) = (2.0 +/- 0.1) x 10(-4) s(-1), pH 7.4, 37 degrees C, [NOMel] = 0.1 mM, [TEMPO] = 9 mM). We also found that NOMel can transnitrosate to l-cysteinate, producing S-nitrosocysteine and melatonin (k = 0.127 +/- 0.002 M(-1) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C). The reaction of NOMel with ascorbic acid as a reducing agent has also been studied. This rapid reaction produces nitric oxide and melatonin. The saturation of the observed rate constant (k = (1.08 +/- 0.04) x 10(-3) s(-1), Tris-HCl (0.2 M) buffer, pH 7.4 at 37 degrees C) at high ascorbic acid concentration (100-fold with respect to NOMel) and the pH independence of this reaction in the pH range 7-9 indicate that the reactive species are ascorbate and melatonyl radical originated from the reversible homolysis of NOMel. Taking into account kinetic and DFT calculation data, a comprehensive mechanism for the denitrosation of NOMel is proposed. On the basis of our kinetics results, we conclude that under physiological conditions NOMel mainly reacts with endogenous reducing agents (such as ascorbic acid), producing nitric oxide and melatonin.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

The isolation of basic proteins by solid-phase extraction with multiwalled carbon nanotubes

Multiwalled carbon nanotubes (MWCNTs) have been employed for the first time as sorbents for the isolation of basic proteins from other protein species in biological sample matrices by solid-phase extraction (SPE). A microcolumn packed with MWCNTs was incorporated after appropriate pretreatment into a sequential injection system, which facilitates online selective sorption of basic protein species (hemoglobin and cytochrome c in this particular case). The retained protein species were afterwards separated from each other by sequential elution from the microcolumn through the employment of appropriate eluents. A 0.025 mol L(-1) phosphate buffer solution of pH 8.0 facilitated the efficient collection of hemoglobin, while a 0.5 mol L(-1) NaCl solution ensured the quantitative recovery of the retained cytochrome c. With a sample loading volume of 2.0 mL, enrichment factors of 11 and 15 were derived for hemoglobin and cytochrome c, along with retention efficiencies of 100% for both species and recovery rates of 98 and 90%, respectively. A sampling frequency of 8 h(-1) was achieved, and the precisions were 3.0% and 0.8% (RSD) for hemoglobin and cytochrome c at a concentration of 5.0 microg mL(-1). The practical applicability of this system was demonstrated by processing of human whole blood for isolation of hemoglobin, and satisfactory results were obtained by assay with SDS-PAGE.     

Fiber optic multi-channel protein detector for use in preparative continuous annular chromatography

On-line coupling continuous-flow liquid membrane extraction (CFLME) with HPLC, a novel automatic system was developed for the determination of sulfonylurea herbicides in water. After an automatic trace-enrichment process by CFLME, which is the combination of continuous flow liquid-liquid extraction and support liquid membrane (SLM) extraction, the target analytes were concentrated in 50 microl of 0.2 M Na2CO3-NaHCO3 (pH 10.0) buffer. The concentrated sample solutions were injected directly onto a C18 analytical column with a valve, and detected at 240 nm with a diode array detector. Metsulfuron methyl (MSM), and DPX-A 7881 were baseline separated with a mobile phase consisting of methanol and 67 mM KH2PO4-Na2HPO4 (pH 5.91) buffer (45+55, v+v) at a flow-rate of 1.0 ml min(-1). With an enrichment time of 10 min and enrichment sample volume of 20 ml, the enrichment factors and detection limits are 100 and 0.05 microg l(-1) for MSM, and 96 and 0.1 microg l(-1) for DPX-A 7881, respectively. The linear range and precision (RSD) are 0.1-50 microg l(-1) and 7.0% for MSM, and 0.2-50 microg l(-1) and 9.2% for DPX-A 7881, respectively. This proposed method was applied to determine MSM and DPX-A 7881 in seawater, tap water, and bottled mineral water with spiked recoveries in the range of 83-95% for MSM and 88-100% for DPX-A 7881, respectively.     

Capillary electrophoresis for analyzing pesticides in fruits and vegetables using solid-phase extraction and stir-bar sorptive extraction

Two procedures based on solid-phase extraction (SPE) and stir-bar sorptive extraction (SBSE) in combination with micellar electrokinetic chromatography (MEKC)--diode array detection (DAD) were compared for the simultaneous extraction of acrinathrin, bitertanol, cyproconazole, fludioxonil, flutriafol, myclobutanil, pyriproxyfen, and tebuconazole in lettuce, tomato, grape, and strawberry. Selectivity and resolution of the MEKC procedure were studied changing the pH and the molarity of the buffer, the type and the concentration of surfactant, and the methanol content in the mobile phase. A buffer consisting of 6 mM sodium tetraborate decahydrate with 75 mM of cholic acid sodium solution (pH 9.2) gave the best results. Linearity, extraction efficiencies and limits of quantitation (LOQs) of both extraction methods were compared. The recoveries obtained by SPE ranged from 40 to 106% with relative standard deviations (R.S.D.s) from 10 to 19% whereas by the SBSE method, the recoveries were 12-47% and the R.S.D.s 3-17%. The LOQs were much better by SPE (0.2-0.5 mg kg(-1) depending on the processed sample amount) than those obtained by SBSE (1 mg kg(-1) for each compound). Advantages and disadvantages of both procedures are also discussed. As SPE is more robust, rapid, and sensitive than SBSE, its application in combination with MEKC is recommended because provided LOQs below the MRLs established, which is not always attained by SBSE.     

固相萃取/高效液相色谱法对肝癌细胞中多西紫杉醇的测定

建立了固相萃取/高效液相色谱法测定肝癌细胞中多西紫杉醇浓度的方法。细胞样品经三氯乙酸沉淀蛋白,Bond-elut C18固相萃取柱提取,采用Agilent TC-C18(5μm,4.6 mm×150 mm)色谱柱分析,以乙腈-0.02 mol/L醋酸铵缓冲液(体积比50∶50,醋酸调至pH5.0)为流动相,流速为1 mL/min,检测波长为230nm,进样体积为20μL。多西紫杉醇的质量浓度在0.05~2.25 mg/L范围内线性关系良好,相关系数r=0.999 3,检出限为0.03 mg/L,提取回收率为93%~95%,其日内、日间精密度(n=5)不大于6.9%,且稳定性良好。方法简便、快速、灵敏度高、干扰小,可用于生物样品中低浓度多西紫杉醇的测定。