On-line turbulent-flow chromatography-high-performance liquid chromatography-mass spectrometry for fast sample preparation and quantitation

A sensitive and selective liquid chromatography-mass spectrometry method has been developed for the simultaneous identification and quantitation of drug substances and metabolites in rat plasma. The method combines on-line turbulent-flow chromatography, high-performance liquid chromatography and mass spectrometry. This combination is considered to be a new approach suitable for fast bio-analysis in drug discovery. Dextromethorphan, and its two metabolites, dextrorphan and 3-methoxymorphinan served as model substances. The analytes present in plasma were collected on a Cyclone column using turbulent-flow chromatography and were subsequently transferred on-line to and focused on an X-Terra MS C8 column. The analytes were eluted by a linear gradient and detected by a fast scanning mass spectrometer. The detector response was quadratic and the dynamic range was estimated to be 0.5-100 ng/ml plasma or 12.5 pg to 2.50 ng injected into the system.     

Analysis of amlodipine in human plasma by liquid chromatography-mass spectrometry

A simple, sensitive, and specific liquid chromatographic method coupled with electrospray ionization (ESI)-mass spectrometry for the determination of amlodipine is developed. After extraction by ethyl acetate using nicardipine as the internal standard, solutes are separated on a C18 column with a mobile phase of methanol-1% HAc (65:35). Detection is performed on an air pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operated in positive-ionization mode. Amlodipine quantitation is realized by computing the peak-area ratio (amlodipine-nicardipine) of the extracts analyzed in single ion monitoring mode (m/z 431 and m/z 480 for amlodipine and nicardipine, respectively) and comparing them with the calibration curve (r = 0.9991).     

Quantitative determination of alpha-, beta-, gamma- and delta-tocopherols in human serum by high-performance liquid chromatography and gas chromatography-mass spectrometry as trimethylsilyl derivatives with a two-step sample preparation

Using a two-step sample preparation with Extrelut and silica gel extraction in Pasteur pipettes it is possible to quantify all tocopherols in human serum samples by means of normal-phase HPLC with fluorescence detection (lambda(ex) 295 nm, lambda(em) 330 nm) or by GC-MS of their trimethylsilyl (TMS) derivatives. The method has been used in pharmacoepidemiological studies concerning the exposition with vitamin E-containing drugs in Germany. The recovery for all tocopherols is 98% and the limit of detection is 50 pg for alpha-tocopherol in the HPLC and 40 pg for all TMS-tocopherols in the GC-MS method using the selected ion monitoring mode with a well-tuned GCQ system. Linearity of calibration is excellent for both methods over the full physiological relevant range. Due to the low sample amount needed, the method is suitable for epidemiological and paediatric research.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

Direct analysis of the major human seminal prostaglandins by thermospray high-performance liquid chromatography-mass spectrometry

Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) can be a powerful tool for characterizing eicosanoids in complex biological samples. The positive ion spectra obtained from primary prostaglandins such as PGE1 PGE2, 19-OHPGE1, 19-OHPGE2, PGF2 alpha, PGD2, 6-keto-PGF1 alpha and from leukotriene B4 are very simple, with base peaks corresponding to ions arising from the loss of H2O from the (M + H)+ and (M + NH4)+ ions, except for PGB2 and PGF2, where the latter two ions predominate. The application of this technique to the concurrent determination of the E1 and E2 prostaglandins and their 19-hydroxylated derivatives in human semen is described. The technique affords a moderate level of sensitivity (5-20 ng on-column) and excellent specificity so that virtually no sample manipulation is required other than dilution in acetone and centrifugation. The clear supernatant is injected directly into the HPLC-MS system. A similar analysis by either gas chromatography (GC) or GC-MS would need multi-step derivatization, thus increasing the sample manipulation required and the total analysis time.     

Proteomic analysis of microvesicles in human saliva by gel electrophoresis with liquid chromatography-mass spectrometry

Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in various ways. They play an important role in tumor progression/metastasis and angiogenesis in cancer and may be useful therapeutic tools, as well as a mechanism of cell-to-cell communication. They have been visioned as an important biomarker or biomarker source for the detection of different diseases. Human saliva is a biological fluid with enormous diagnostic potential, which harbors plenty of salivary MVs. The goal of this study is to investigate the proteomic profiling of MVs in human saliva through a simple preparation procedure by using filtration and centrifugation. Gel electrophoresis was combined with LC–MS/MS (liquid chromatography–mass spectrometry) for the proteomic analysis of MVs. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein separation, the whole lane was cut into 25 bands, and each band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC–MS/MS for protein identification. Through protein database search, 63 proteins were identified for human salivary MVs. Several members of different protein families were identified, including annexin, keratin, actin, immunoglobulin and S100. This study showed that although there was an overlap with the proteins from human saliva and salivary exosomes, salivary MVs contained their own unique proteins. These results will poise human salivary MVs as a non-invasive tool for the early detection of different diseases.     

Determination of telenzepine in human serum by gas chromatography-mass spectrometry

A method for determining telenzepine in human serum is described. Analytes are obtained from alkalinized serum by extraction of the drug using reversed-phase octadecylsilane-bonded silica cartridges. Telenzepine and a desmethyl analogue added to serum as internal standard are retained on the C18 cartridge and recovered by elution with methanol. The gas chromatographic properties of telenzepine and the internal standard are improved by a two-step derivatization involving a benzodiazepinone-benzimidazole rearrangement and simultaneous formation of a methyl ester function. The processed extract is analysed by gas chromatography-mass spectrometry with selected-ion monitoring. Quantification is linear over the range 2-40 ng/ml. Inter-day precision is within 7%, except at the detection limit of 2 ng/ml (16%). Application of this assay to routine analysis is limited by the extensive sample pretreatment essential for derivatization of telenzepine.     

Sequence analysis of derivatized peptides by high-performance liquid chromatography-mass spectrometry

N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.     

Enantioselective analysis of methadone in saliva by liquid chromatography-mass spectrometry

Saliva was tested and evaluated as a biological matrix for methadone (Mtd) monitoring. Conventional method using a narrow bore C18 column, and an enantioselective method using a narrow bore alpha1-acid glycoprotein column, were developed using liquid chromatography coupled with a mass spectromeric (MS) detector. After optimisation of MS conditions by flow injection analysis, selected ion monitoring detection was used to enhance sensitivity. The total Mtd concentration and the enantiomeric ratio in saliva were validated using an experimental design. The methods were applied to samples provided by heroin addicts undergoing a Mtd treatment. Results on total Mtd determination showed a very poor correlation between saliva and serum, whereas the enantiomeric ratios of Mtd gave a very good one.     

Automated sample preparation on-line with thermospray high-performance liquid chromatography-mass spectrometry for the determination of drugs in plasma

The combination of a solid-phase extraction module, the AASP, on-line with thermospray high-performance liquid chromatography-mass spectrometry for the automated determination of drugs in plasma is described. The technique was evaluated successfully using, as an example, the determination of labetalol in human plasma. [2H7]Labetalol was used as an internal standard to compensate for changes in ionization efficiencies between analyses. The chromatographic and mass spectrometric conditions were optimized for labetalol. The combined technique was demonstrated as being robust and reliable for the analysis of plasma samples from a clinical study.     

Metabolite identification by liquid chromatography-mass spectrometry

Metabolite identification (Met ID) is important during the early stages of drug discovery and development, as the metabolic products may be pharmacologically active or toxic in nature. Liquid chromatography-mass spectrometry (LC-MS) has a towering role in metabolism research.This review discusses current approaches and recent advances in using LC-MS for Met ID. We critically assess and compare various mass spectrometers, highlighting their strengths and limitations. Citing appropriate examples, we cover recent LC and ion sources, isotopic-pattern matching, hydrogen/deuterium-exchange MS, data dependent analyses, MS^E, mass defect filter, 2D and 3D approaches for the elucidation of molecular formula, polarity switching, and background-subtraction and noise-reduction algorithms. A flow chart outlines a comprehensive strategy for Met ID, including a focus on reactive metabolites.     

Analysis of DNA adducts of acetaldehyde by liquid chromatography-mass spectrometry

A highly sensitive method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was developed for the analysis of DNA adducts of acetaldehyde (AA). AA, which is the primary oxidative metabolite of ethanol, is considered to possess carcinogenic activity. AA reacts with the exocyclic amino group of guanine in DNA to form N2-ethylguanine (Et-Gua) and 1,N2-propanoguanine (Pr-Gua) adducts. With the present method, such adducts were detected as the base forms from DNA chains using depurination in the pretreatment process. In our measurement with LC-ESI-MS, the limits of detection (LODs) of the Et-Gua and Pr-Gua adducts of the base forms were 3.0 x 10(-10) and 1.0 x 10(-9) M, respectively, and the LODs are about two orders of magnitude lower than those of the nucleoside forms. Calf thymus DNA samples treated with AA and NaBH3CN were analyzed by this method. Et-Gua was clearly detected and, in the absence of NaBH3CN, Pr-Gua was detected predominantly. Furthermore, the method was also applied to study whether or not these two adducts are formed in DNA of cultured HL-60 cells during exposure to AA for 24 h. Pr-Gua was clearly detected and traces of Et-Gua were also detected in the DNA of the cells. Although the sensitivity of this method is lower by at least oneorder of magnitude than the 32P-postlabeling assay, currently the most sensitive method, our method does not involve complex enzymatic reactions for the postlabeling and the use of troublesome radioactive materials. Furthermore, it enables structural identification of guanine adducts. The present method would be a useful tool for studies of Et-Gua and Pr-Gua adducts in connection with carcinogenesis.     

Matrix effects in pesticide multi-residue analysis by liquid chromatography-mass spectrometry

Three sample preparation methods: Luke method (AOAC 985.22), QuEChERS (quick, easy, cheap, effective, rugged and safe) and matrix solid-phase dispersion (MSPD) were applied to different fruits and vegetables for analysis of 14 pesticide residues by high-performance liquid chromatography with electrospray ionization-mass spectrometry (HPLC/ESI/MS). Matrix effect, recovery and process efficiency of the sample preparation methods applied to different fruits and vegetables were compared. The Luke method was found to produce least matrix effect. On an average the best recoveries were obtained with the QuEChERS method. MSPD gave unsatisfactory recoveries for some basic pesticide residues. Comparison of matrix effects for different apple varieties showed high variability for some residues. It was demonstrated that the amount of co-extracting compounds that cause ionization suppression of aldicarb depends on the apple variety as well as on the sample preparation method employed.     

Analysis of cyanobacterial toxins by hydrophilic interaction liquid chromatography-mass spectrometry

The combination of hydrophilic interaction liquid chromatography with electrospray mass spectrometry (HILIC-MS) has been investigated as a tool for the analysis of assorted toxins produced by cyanobacteria. Toxins examined included saxitoxin and its various analogues (1-18), anatoxin-a (ATX-a, 19), cylindrospermopsin (CYN, 20), deoxycylindrospermopsin (doCYN, 21), and microcystins-LR (22) and -RR (23). The saxitoxins could be unequivocally detected in one isocratic analysis using a TSK gel Amide-80 column eluted with 65% B, where eluent A is water and B is a 95% acetonitrile/water solution, both containing 2.0 mM ammonium formate and 3.6 mM formic acid. The analysis of ATX-a, CYN and doCYN required 75% B isocratic. Simultaneous determination of 1-21 was also possible by using gradient elution. HILIC proved to be suitable for the analysis of microcystins, but peak shape was not symmetric and it was concluded that these compounds are best analysed using existing reversed-phase methods. The HILIC-MS method was applied to the analysis of field and cultured samples of Anabaena circinalis and Cylindrospermopsis raciborskii. In general, the method proved quite robust with similar results obtained in two different laboratories using different instrumentation.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

Monitoring of cefepime in human serum and plasma by micellar electrokinetic capillary chromatography: Improvement of sample preparation and validation by liquid chromatography coupled to mass spectrometry

Cefepime monitoring in deproteinized human serum and plasma by micellar electrokinetic capillary chromatography and liquid chromatography coupled to mass spectrometry in presence of other drugs is reported. For micellar electrokinetic capillary chromatography, sample preparation comprised dodecylsulfate protein precipitation at pH 4.5 using an increased buffer concentration compared to that of a previous assay and removal of hydrophobic compounds with dichloromethane. This provided robust conditions for cefepime analysis in the presence of sulfamethoxazole and thus enabled its determination in samples of patients that receive cotrimoxazole. The liquid chromatography assay is based upon use of a column with a pentafluorophenyl‐propyl modified and multiendcapped stationary phase and the coupling to electrospray ionization with a single quadrupole detector. The performances of both assays with multilevel internal calibration were assessed with calibration and control samples and both assays were determined to be robust. Cefepime levels monitored by micellar electrokinetic capillary chromatography in samples from patients that were treated with cefepime only and with cefepime and cotrimoxazole were found to compare well with those obtained by liquid chromatography coupled to mass spectrometry. Cefepime drug levels determined by micellar electrokinetic capillary chromatography could thereby be validated.     

Quantitative determination of tramadol in human serum by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the quantitative determination of tramadol in human serum, plasma or whole blood samples is described. The method involves the use of [2H2, 15N]tramadol hydrochloride as an internal standard and chemical ionization with isobutane, employing single-ion monitoring for quantification. It is specific, sensitive and precise, and has high accuracy. The within-run coefficient of variation is about 1% between 25 and 200 ng/ml and 1.8-2.9% at the lowest concentrations tested (6.25 and 12.5 ng/ml). The between-run coefficient of variation increases from 1.6% to 5.2% with decreasing concentration from 200 to 12.5 ng/ml. The calibration graphs were linear in the tested concentration range, and the accuracy of the assay was not dependent on the sample volume used. The detection limit was about 4 ng/ml for serum samples of 1 ml. The method proved suitable for pharmacokinetic studies. Its high sensitivity allows measurements of serum concentrations for at least 30 h after the single administration of therapeutic doses of tramadol hydrochloride.     

Automated method for targeting analysis of prostanoids in human serum by on-line solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring

Prostanoids are potent biologically active lipid molecules demanding for analysis methods combining precision, sensitivity and high-throughput for pharmacological and clinical applications. The present research describes the development and validation of an on-line automated method based on solid-phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for the quantification of prostanoids in human serum. This approach overcomes the main limitation of previous methods involving manual protocols, such as analyte losses, metabolites degradation and time-consuming protocols, are minimized. Human serum (100 μL) was directly injected into an automatic solid-phase extraction workstation for cleanup and preconcentration of the target metabolites. The eluate was on-line transferred to a reversed-phase analytical column for chromatographic separation prior to mass spectrometry detection in selected reaction monitoring mode. The detection limits for the target analytes ranged from 2.3 to 63.3 pg on column. The precision (expressed as relative standard deviation) was within 3.30 and 6.15% for repeatability and from 4.16 to 11.11% for within-laboratory reproducibility. Accuracy was evaluated with spiked and non-spiked serum samples to estimate concentration differences that could be affected by matrix effects or inefficient SPE performance. Accuracy, estimated as recovery factor, was from 87.7 to 100% for the target compounds. The proposed method is reliable and has an excellent potential for high-throughput use in both clinical and research laboratories by minimizing analyst intervention.