Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns

A novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) method is described for the determination of vancomycin in serum and urine. After the addition of internal standard (teicoplanin), serum and urine samples were directly injected onto an HPLC system consisting of an extraction column and dual analytical columns. The columns are plumbed through two switching valves. A six-port valve directs extraction column effluent either to waste or to an analytical column. A ten-port valve simultaneously permits equilibration of one analytical column while the other is used for sample analysis. Thus, off-line analytical column equilibration time does not require mass spectrometer time, freeing the detector for increased sample throughput. The on-line sample extraction step takes 15 seconds followed by gradient chromatography taking another 90 seconds. Having minimal sample pretreatment the method is both simple and fast. This system has been used to successfully develop a validated positive-ion electrospray bioanalytical method for the quantitation of vancomycin. Detection of vancomycin was accurate and precise, with a limit of detection of 1 ng/mL in serum and urine. The calibration curves for vancomycin in rat, dog and primate were linear in a concentration range of 0.001-10 microg/mL for serum and urine. This method has been successfully applied to determine the concentration of vancomycin in rat, dog and primate serum and urine samples from pharmacokinetic and urinary excretion studies.     

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.     

Direct analytical and preparative resolution of enantiomers using albumin adsorbed to silica as a stationary phase

A rapid and simple method for the preparation of high-performance liquid chromatography columns for chiral separations is described. The stationary phase is prepared by adsorbing bovine serum albumin on silica. Both analytical and preparative applications are described. A polarimeter was used as a detector to determine the enantiomer elution orders.     

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

适用于质谱分析的在线固相萃取除盐方法

建立了一个简单、快速、有效的适用于质谱或液相色谱-质谱联用的在线固相萃取(SPE)高通量除盐方法。方法分为单柱和双柱模式,借助于包含双梯度泵(上样泵/分析泵)、自动进样器和配有十通切换阀的柱温箱的高效液相色谱系统,完成样品的自动化在线除盐。单柱模式通过上样泵实现在SPE柱上进样和除盐,被分析物则保留在SPE柱上;除盐完成后,通过阀切换利用分析泵洗脱富集在SPE柱上的被分析物。双柱模式则在单柱模式基础上增加了1根SPE柱,在色谱管理软件控制下2根SPE柱轮流工作,高效率完成样品的在线除盐。该方法在结合质谱分析蛋白质、多肽等领域具有较好的应用前景。     

HPLC determination of cis-diamminedichloroplatinum(II) in plasma and urine with UV detection and column-switching

A method for determining cis-diamminedichloroplatinum(II) (CDDP), an anticancer drug, in plasma and urine by HPLC with UV detection and column-switching has been developed. Typical conditions were as follows. An apparatus was composed of two columns, two pumps, a UV detector, a sample injector with a 100 microL loop, a switching valve, a column oven and a recorder. A Rheodyne model 7125 sample injector was used as the switching valve. A precolumn (4.6 mm ID x 25 cm) was packed with MCI GEL CK10S (a strong cation exchanger), and an analytical column (4.6 mm ID x 5 cm) was packed with MCI GEL CDR10 (a strong anion exchanger). Both columns were connected in series via the switching valve. The CDDP-containing fraction of the effluent from the precolumn was loaded to the analytical column by column-switching and the effluent from the analytical column was monitored at 210 nm. An eluent of 0.3 M sodium dihydrogen phosphate was pumped at a flow rate of 1 mL/min and the columns were maintained at 40 degrees C. CDDP was eluted at about 11 min and the identity of the peak of CDDP on the chromatogram was confirmed by its 3-dimensional chromatogram and analysis of platinum in the column effluent. Under the conditions described above, a linear relationship was obtained between peak height and concentration of CDDP up to 100 microM. Correlation efficients were 0.998 for plasma and 0.999 for urine. The detection limit was 0.1 microM for CDDP in both plasma and urine (S/N = 3,0.005 AUFS). The reproducibility was within 3% for 10 determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Rapid liquid chromatography for paralytic shellfish toxin analysis using superficially porous chromatography with AOAC Official Method 2005.06

The bioaccumulation of paralytic shellfish toxins in mussels, oysters, cockles, hard clams, razors, and king scallops is monitored in England, Scotland, and Wales by AOAC Official Method 2005.06 LC-with fluorescence detection (FLD). One of the commonly perceived disadvantages of using this method is the long turnaround time and low throughput in a busy laboratory environment. The chromatographic analysis of each sample typically utilizes a 15 min cycle time to achieve toxin oxidation product separation and column equilibration prior to subsequent analysis. A standard RP C18 analytical column, used successfully in recent years, achieves good separation with a long column lifetime. The analysis of a 40 sample qualitative screening batch takes approximately 18 h, including blanks, standards, and other QC samples. The availability of superficially porous column technology has offered the potential to reduce analysis time while retaining column performance on existing hardware. In this study, AOAC Official Method 2005.06 with LC-FLD was transferred to two different commercially available superficially porous columns, and the method performance characteristics were evaluated. Both columns separated all toxins adequately with cycle times less than half that of the existing method. Linearity for each toxin was acceptable up to two times the European maximum permitted limit of 800 microg di-HCl saxitoxin equivalent/kg flesh. LOD and LOQ values were substantially improved for the majority of toxins, with gonyautoxin 1&4 and neosaxitoxin showing up to a two- and fourfold improvement, respectively, depending on the column used. Quantification results obtained from parallel analysis of contaminated samples were acceptable on both columns. Comparative screen results gave a slight increase in the occurrence of contaminated samples, which was attributed to the improved detection limit for most toxins. Issues with rapidly increasing back pressure, however, were identified with both columns, with a limit of around 500 injections. This compares to the >3000 cycles routinely obtained with the standard RP-C18 HPLC columns currently in use. Overall, the gain achieved with these columns through shorter analysis time and improved analytical sensitivity is potentially of benefit in a high-throughput environment. For the routine high-throughput screening of shellfish samples, however, an improved column lifetime is desirable.     

Sample clean-up by sol-gel immunoaffinity chromatography for determination of chloramphenicol in shrimp

The paper describes the characterisation and application of sol-gel columns prepared by entrapping anti-chloramphenicol (CAP) antibodies. Retention of CAP in the column was caused by specific interactions with the anti-CAP antibodies and not by non-specific adsorption to the sol-gel glass. After optimising important operation conditions, e.g. feeding medium, feeding flow-rate, elution medium, elution flow-rate and elution volume, the sol-gel columns were included in a clean-up procedure developed to determine CAP in shrimp. The selectivity of the columns was high enough to efficiently remove interfering matrix compounds. Due to the chromatographic conditions applied retention of cross-reacting substances in the immunoaffinity column did not pose a problem. CAP recovery of the analytical method was 68% with a relative standard deviation of 4% (n=4). In spite of applying highly complex shrimp extracts the columns could be used for clean-up of at least 12 samples. However, when detection of CAP is carried out with an UV detector the analytical method has a relatively poor sensitivity (LOD=1.8 ng/g, S/N=3). The most obvious way is to replace the UV detector by a detector based on an inherently more sensitive and selective detection principle, like a mass spectrometer.     

Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid-ethanol fluorescence derivatization and column switching.

An assay method for serum cortisol, using precolumn sulphuric acid-ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence intensity increased. Among the sulphuric acid-ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 micrograms/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.     

Determination of trichloroacetate in human serum and urine by ion chromatography

A simple and rapid ion chromatographic method has been developed for the determination of trichloroacetate in human serum and urine. Two short (50 x 5 mm i.d.) low-capacity anion-exchange resin columns in series were used as the analytical column and a mixture of 35 mM sodium hydroxide, 20 mM p-hydroxybenzonitrile and 2% V/V acetonitrile (pH 12.3) as the eluent. The calibration graph was rectilinear from 0.3 to 33 mg l-1 of trichloroacetate at 10 microS with a detection limit (3 sigma) of 0.08 mg l-1. The recovery of trichloroacetate, obtained by analysing spiked serum and urine samples, was in the range 98.2-101% and the coefficient of variation was always less than 3.0%.     

Two-dimensional reverse phase-reverse phase chromatography: A simple and robust platform for sensitive quantitative analysis of peptides by LC/MS. Hardware design

We have revised current two-dimensional RP-RP approaches and developed a new robust 2-D RP-RP platform. This platform was implemented on an Agilent 1100 2-D liquid chromatography system and is based on high pressure switching between two high-resolution RP columns. An independent binary gradient was implemented for each dimension. The powerful combination of dual analytical columns with independent gradient elution achieves high analyte purity, effectively eliminates matrix effects, and maximizes MS sensitivity in Q1 SIM comparable to the sensitivity enhancements of MS/MS-based methods. Implementation of dual simultaneous gradient profiles (overlapped gradients) reduces 2-D method run-time to the scale of 1-D method run-times. This robust and sensitive approach is particularly suitable for hydrophobic peptides and small proteins and can be used as a routine standard technique for enhanced on-line peptide purification coupled with mass spectrometric detection.     

Comparison of sub-2-microm particle columns for fast metabolite ID

The use of sub-2-microm particle columns for fast high throughput metabolite ID applications was investigated. Three LC-MS methods based on different sub-2-microm particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 microm particle column coupled to an MS, methods B and C utilized a 1.7 microm particle column (BEH 50 x 2.1 mm2 id) and 1.8 microm particle column coupled to a Q-TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N=10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O-demethylverapamil (ODMVP) in human-liver microsomal incubates. Results show that 1.8 microm particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MS(E) mode of the Q-TOF instrument compared to 1.7 microm particle columns. Additionally, the study also confirmed that sub-2-microm particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384-well plate format of up to 48 compounds in incubates of human-liver microsomes was discussed.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.     

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.     

Column-switching techniques for high-performance liquid chromatography of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection.

Column-switching techniques for high-performance liquid chromatography of two acidic drugs, ibuprofen and mefenamic acid, in human serum with short-wavelength ultraviolet detection are described. The method involved extraction of the analyte from acidified serum followed by the chromatographic analysis using column switching. Three ODS columns were used each with different mobile phase, utilizing the difference of ion-pair formation or of ionization caused by pH change. The method offered high sensitivity and selectivity, with short-wavelength ultraviolet detection at 221 nm for ibuprofen and at 219 nm for mefenamic acid. The detection limits were 0.5 ng/ml (2.4 pmol/ml) for ibuprofen and 0.1 ng/ml (0.4 pmol/ml) for mefenamic acid using 1 ml of serum, both at a signal-to-noise ratio of 3. With some modifications, the principle of the method would be applicable to other acidic compounds in biological fluids.     

High throughput on-line solid phase extraction/tandem mass spectrometric determination of paclitaxel in human serum

The feasibility of high throughput on-line solid phase extraction/tandem mass spectrometry (SPE/MS/MS) is tested for target analysis of paclitaxel in human serum. The use of a dual Prospekt system, with parallel SPE and elution directly to the mass spectrometer, resulted in a cycle time of 80 seconds for the entire, fully automated assay. The assay proved to be linear from 1 to 1000 ng/mL. Cartridges packed with small sorbent particles functioned both as SPE cartridges and as short analytical columns.     

多壁碳纳米管固相萃取净化气相色谱法分析蔬菜中有机氯和拟除虫菊酯农药残留

建立了多壁碳纳米管为吸附剂的固相萃取(SPE)净化、气相色谱-电子捕获检测(GC-ECD)测定蔬菜中6种有机氯和7种拟除虫菊酯农药的方法。采用双柱(HP-50和HP-1色谱柱)双检测器进行定性和定量分析。蔬菜样品采用乙腈提取,多壁碳纳米管SPE柱净化,正己烷溶解上样,丙酮-正己烷(7:3, v/v)洗脱,13种农药中有11种农药的添加回收率高于70%。将该净化方法用于荷兰黄瓜、卷心菜、红圣女果、奶油生菜、紫甘蓝、韭菜、大葱和洋葱等样品的净化,与弗罗里硅土SPE柱相比较,净化效果更好,表明多壁碳纳米管具有较强的吸附去除色素的能力,可以避免色素对测定的干扰。     

Screening of pesticides via solid-phase extraction and gas chromatography

This work describes an analytical method for the screening of pesticide residues in water. The developed method identifies and quantitates a variety of pesticides in water samples such as organochlorine, organophosphate, chloronitrile, phthalimide, dicarboximide, and triazine. The analytical technique employed for the extraction and cleanup step involves solid-phase extraction with C-18 cartridges. The identification and determination of concentration is carried out with gas chromatography. Two columns of different polarity and two specific detectors are used. An electron capture detector is used for organochrolorine, and a thermoinic detector is used for organonitrogen and organophosphate. The good resolution achieved with the temperature program and the combination of columns is shown in the chromatograms obtained. The limits of detection, percent recovery, and their respective standard deviations are determined for each product. According to the results obtained, this method permits monitoring of the pesticides mentioned, many of which are moderately to highly toxic to aquatic organisms.