Solid-phase Sample Preparation Research Articles

Toward a Universal Sample Preparation Method for Denaturing Top-Down Proteomics of Complex Proteomes.

A universal and standardized sample preparation method becomes vital for denaturing top-down proteomics (dTDP) to advance the scale and accuracy of proteoform delineation in complex biological systems. It needs to have high protein recovery, minimum bias, good reproducibility, and compatibility with downstream mass spectrometry (MS) analysis. Here, we employed a lysis buffer containing sodium dodecyl sulfate for extracting proteoforms from cells and, for the first time, compared membrane ultrafiltration (MU), chloroform-methanol precipitation (CMP), and single-spot solid-phase sample preparation using magnetic beads (SP3) for proteoform cleanup for dTDP. The MU method outperformed CMP and SP3 methods, resulting in high and reproducible protein recovery from both Escherichia coli cell (59 ± 3%) and human HepG2 cell (86 ± 5%) samples without a significant bias. Single-shot capillary zone electrophoresis (CZE)-MS/MS analyses of the prepared E. coli and HepG2 cell samples using the MU method identified 821 and 516 proteoforms, respectively. Nearly 30 and 50% of the identified E. coli and HepG2 proteins are membrane proteins. CZE-MS/MS identified 94 histone proteoforms from the HepG2 sample with various post-translational modifications, including acetylation, methylation, and phosphorylation. Our results suggest that combining the SDS-based protein extraction and the MU-based protein cleanup could be a universal sample preparation method for dTDP. The MS raw data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD018248.

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis.

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96-well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on-bead digested by using Single-Pot solid-phase sample preparation (SP3). The whole IP-SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter-aided sample preparation) or a longer incubation protocol. Taken together, the IP-SP3 protocol is a fast and economical approach easily applicable for large-scale protein interactome analysis.

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)–PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for β-thalassemia.

High-performance liquid chromatographic analysis of nitrendipine in human plasma using ultraviolet detection and single-step solid-phase sample preparation

A high-performance liquid chromatographic (HPLC) method was developed for the determination of nitrendipine in human plasma using solid-phase extraction (SPE) and ultraviolet detection. A 30-μl aliquot of methanol (containing 2 μg/ml of the internal standard, nimodipine) was added to a 1-ml aliquot of biological sample. After vortex-mixing, the mixture was loaded on C 18 SPE cartridge which was conditioned with methanol and distilled water. After washing with distilled water, the SPE cartridge was eluted with 1-ml aliquot of diethyl ether. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-μl aliquot of mobile phase, and a 50-μl aliquot was injected onto the C 18 reverse-phased column. The mobile phase, 10 mM phosphate buffer (pH 4.5):acetonitrile (50:50, v/v), was run at a flow rate of 1.2 ml/min. The column effluent was monitored using ultraviolet detector at 238 nm. The retention times for nitrendipine and the internal standard were approximately 10.1 and 12.6 min, respectively. The detection limit of nitrendipine in human plasma was 2.0 ng/ml. The coefficients of variation (CV) of the assay were below 16.5% for human plasma, and no interferences from endogenous substances were found. This specific, accurate and precise assay was useful in the study for the pharmacokinetic characteristics of nitrendipine.

Liquid chromatographic–tandem mass spectrometric method for the analysis of a neurokinin-1 antagonist and its metabolite using automated solid-phase sample preparation and automated data handling and reporting

Pharmacokinetic studies play a vital role during the development of new pharmaceutical substances. Data presented demonstrate an accurate, precise and robust assay for a neurokinin-1 receptor antagonist and its metabolite with HPLC-MS-MS. Sample preparation is performed by solid-phase extraction (SPE) in the 96-well plate format. This process is fully automated with a Tecan Genesis™ pipetting system using its standard robotic manipulator arm (ROMA). All instruments are fully integrated in a study oriented laboratory information system (LIMS) with an Oracle database that communicates bi-directional with the analytical equipment. Finally, the results are reported by push button operation.

Quantitative analysis of sirolimus (Rapamycin) in blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

We report here a quantitative method for the analysis of sirolimus in blood using solid-phase sample preparation and HPLC–electrospray-tandem mass spectrometry detection. Blood samples (500 μl) were prepared by pre-treatment with acetonitrile: 15 m M zinc sulphate (70:30, v/v), containing 32-demethoxysirolimus (internal standard) and C 18 solid-phase extraction. The electrospray conditions were chosen to enhance the [M+NH 4] + species at the expense of other species. Detection was by multiple reactant monitoring with the mass transitions m/z 931.8→864.6 and m/z 901.8→834.4 employed for sirolimus and the internal standard, respectively. The method was linear over the range 0.2 to 100.0 μg l −1. The accuracy and inter-day precision, over this concentration range, was 94.4% to 104.4% and 1.4% to 5.0%, respectively. The accuracy and total precision at the limit of quantitation (0.2 μg l −1) was 103.0% and 10.8%, respectively. The mean absolute recovery of sirolimus and the internal standard were 80.5% and 81.3%, respectively. The sensitivity and analytical concentration range of the method make it suitable for therapeutic drug monitoring and pharmacokinetic studies. Further, the ability of the method to measure parent drug specifically will facilitate the evaluation of immunoassays for sirolimus.

Fluorescence detection of cardenolides in reversed-phase high-performance liquid chromatography after post-column derivatization

Methods for the fluorescence derivatization of cardiac glycosides with concentrated acids from TLC are adopted to HPLC for post-column derivatization. The column effluent is blended with concentrated acids in a knitted tube reactor, which enables derivatization with negligible increase in chromatographic peak width. The selectivity of the reaction is temperature-dependent and influenced by the respective acid. Reactivity increases from H 3PO 4→CH 3SO 3H ≡ H 2SO 4. The conversion of digoxigenin, digitoxigenin and their digitoxosides is accelerated by Cu(II) acetate or Co(II) nitrate in H 2SO 4. Combined with a new two-mode, single-column solid-phase sample preparation, cardiac glycoside levels of less than 100 pg/glycoside in 1 ml plasma are detectable.

Sensitive high-performance liquid chromatographic analysis of amlodipine in human plasma with amperometric detection and a single-step solid-phase sample preparation

A narrow-bore HPLC assay with electrochemical detection for the determination of the calcium antagonist amlodipine in human plasma samples is presented. By using a single-step solid-phase extraction procedure on Bond Elut C 2 columns, the sample preparation step has been considerably simplified and less time-consuming compared to earlier presented works. With a linear and reproducible calibration curve over the range 0.5–20 ng ml −1 plasma, the assay has successfully been used in the analysis of more than 500 plasma samples from a multicenter trial.

Determination of sulfadoxine concentrations in whole blood using C18 solid-phase extraction, sodium dodecyl sulfate and dimethylaminocinnamaldehyde.

A simple method is described for the extraction and subsequent analysis of sulfadoxine in human whole blood using a solid-phase extraction technique and colorimetric reaction. This procedure utilizes the micellar properties of sodium dodecyl sulfate to: (1) extract sulfadoxine from a C18 solid-phase sample-preparation column; (2) enhance the colorimetric reaction produced by the addition of p-dimethylaminocinnamaldehyde (DMAC); and (3) provide stability to the coloured product generated by the reaction of sulfadoxine with DMAC. The intense, violet-red colour reaction can be conveniently used for qualitative and semiquantitative visual interpretations of sulfadoxine levels. Under the assay conditions, drug concentrations in the blood of subjects receiving sulfadoxine were determined from absorbance measurements. These results correlated well with the sulfadoxine levels determined from high-performance liquid chromatographic analysis. Important advantages of the procedure include the ability to evaluate small samples of whole blood (100 microliters), the minimal use of organic solvents, no sophisticated instrumentation, and formation of a stable, coloured reaction product. The method proved to be a suitable field assay for determining whole-blood levels of sulfonamides in the concentration range from 5 to 100 micrograms ml-1.

Solid-phase sample preparation and stability of pesticides in water using Empore disks

The use of C 18 and styrene—divinylbenzene (ST-DVB) Empore extraction disks for trace-enrichment and determination of the stability of various pesticides in water samples is shown. The influence of the water type and of matrix interferences in the determination of pesticides at the 0.02–1 μg/l level is discussed. Impurities generated by the matrix and the sorbent of the disk have been identified by gas chromatography—mass spectrometry (GC-MS) and their concentrations have been compared with impurities found when using conventional extraction cartridges. For the extraction of acidic herbicides the levels of interferences at pH 1 have been investigated. The recoveries obtained with the Empore disks and with conventional liquid—liquid extraction (LLE) are compared. Finally examples of stability studies of pesticides enriched in the disk materials are given.

Solid-phase sample preparation of natural waters with reversed-phase disks

An investigation of sample preparation for natural waters using the Empore disk was conducted. The Empore disk is a new solid-phase sample preparation technology which was developed for rapid isolation of organic contaminants from aqueous matrix. In order to increase the volume of water that could be prepared, it was found that in-line or off-line filtration prior to the extraction step was required. The appropriate filters were identified. When more than 10 liters of natural water were analyzed a non-specific interference to capillary gas chromatography-electron-capture detection determination of analytes was present. The use of the Empore disks offered some advantages in ease and specificity of elution that was not available with solvent extraction and other solid-phase sample preparation technologies.

Determination of reactive nitrogen mustard anticancer drugs in plasma by high-performance liquid chromatography using derivatization

A high-performance liquid chromatography (HPLC) method is described for the determination of reactive nitrogen mustard anticancer drugs in plasma after derivatization with diethyl-dithiocarbamic acid (DDTC). Three compounds were studied: two reactive species (mechlorethamine (HN2) and galactose 6-mustard (G-6-M] and a less reactive species (melphalan (L-PAM] included for validation experiments. Mass and NMR spectrometry confirmed that one molecule of DDTC reacts with each arm of the mustard, displacing a chlorine atom to form a stable disubstituted adduct. With the reactive mustards a 30-min incubation at 37 degrees C is recommended for greater than 90% derivatization efficiency. Gradient elution was employed to analyze all three compounds using the same conditions with a microBondapak C18 10-microns particle size column (30 cm by 3.8 mm i.d.). The retention time (tR) of HN2-DDTC2 was 13.1 min +/- 1.5% within day CV; tR of L-PAM was 7.6 min and L-PAM-DDTC2 was 14.6 min +/- 0.8% CV. G-6-M-DDTC2 yielded a double peak, tR = 10.7 min and 10.9 min +/- 2.9% CV. The limit of detection on column was 0.5 ng for HN2, 1 ng for L-PAM, and 5 ng for G-6-M. A solid-phase sample preparation technique using "Bond Elut" phenyl is described that extracts from plasma G-6-M-DDTC2 with greater than 74% efficiency and HN2-DDTC2 with greater than 90% efficiency. When the drugs were derivatized in plasma, recovery remained high for G-6-M (greater than 84%) but dropped to 50% for HN2.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for the determination of γ- l-glutamyl- l-dihydroxyphenylalanine and its major metabolites l-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the analysis of γ- l-glutamyl- l-dihydroxyphenylalanine (gludopa) and its major metabolites l-dihydroxyphenylalanine ( l-DOPA), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is described. High sensitivity is achieved with a multi-cell coulometric detector utilising the specific electrochemical properties of gludopa (limit of detection 10 pg on-column). The retention time of gludopa was both pH-dependent and sensitive to negatively charged ion-pairing agents. An alumina-based solid-phase sample preparation technique with dihydroxybenzylamine as internal standard is described for plasma and urine (limit of detection 40 pg/ml) and an ultrafiltration technique is described for tissues (limit of detection 1–10 ng/g). After treatment with 50 mg/kg gludopa, in excess of twenty separate catecholic metabolic peaks can be detected in rat urine, whereas in humans after 9 mg/kg the only catechols detected were l-DOPA, dopamine and DOPAC.

A rapid and specific HPLC-electrochemical method for the determination of endogenous 5-methyltetrahydrofolic acid in plasma using solid phase sample preparation with internal standardization.

A rapid and specific HPLC-electrochemical method for determining endogenous 5-methyltetrahydrofolic acid (5MeTHF) in plasma is described. Quantitative solid phase extraction of 5MeTHF and internal standard, beta-hydroxyethyltheophylline, was carried out using proprietary phenyl bonded-silica columns (Bond Elut Phenyl cartridges, 1.0 mL capacity). Chromatographic separation was achieved using a mobile phase consisting of 15% (v/v) methanol in 0.05 M KH2PO4, pH 3.5 at a flow rate of 2.0 mL/min in conjunction with a Waters Assoc. radially compressed Nova-Pak phenyl column (10 cm x 8 mm, 4 microns bonded silica). The internal standard was measured by UV detection at 254 nm. A Bioanalytical Systems Inc. LC-17 glassy carbon oxidative flow cell with a potential held at +0.35 V vs Ag/AgCl using the LC-4A amperometric controller allowed levels of 1-2 ng/mL 5MeTHF to be measured in 500 microL of plasma. Daily appraisal of the ratio produced by authentic materials clearly demonstrated that quantitation using dual detection was not subject to problems of differential response. Inter-day variation of the differential detector response is cited. Comparison of the Lactobacillus casei bioassay with HPLC demonstrates good agreement between methods but at the same time highlights the drawback of using such non-specific methods to measure samples where more than one folylmonoglutamate may be present. Antoxidant free storage for three months at -70 degrees C in darkness resulted in no deterioration of 5MeTHF. A comparison of the means and range of values for plasma folate obtained using HPLC, L. casei bioassay and the radiometric binding assay is reported.

Solid-phase sample preparation method for prostaglandins: integration of procedures for isolation and derivatization for gas chromatographic determination.

A procedure using a solid-phase support has been developed for the isolation and derivatization of prostaglandins from biological matrices. The styrene-divinylbenzene cross-linked copolymeric macroreticular resin, XAD-2, was used as an adsorbent for prostaglandin E2 from biological samples, as a support for the oximation of the carbonyl group and as a catalyst for pentafluorobenzylation. The reactor bed was then linked to a Florisil column for a final chromatographic clean-up. Matrix effects were found to affect the yield, but recovery of the desired electrophoric products was comparable with methods reported in the literature. The ease of sample preparation suggests that this technique may be a viable approach to automating the processes for preparing prostaglandins from biological matrices for gas chromatographic analysis.

Automated and Manual In-Line Solid-Phase Sample Preparation for High-Performance Liquid Chromatography Analysis of Bittering Substances in Worts and Beers

The feasibility of in-line solid-phase treatment of samples (5–50 μl) of stout, ale, and lager beers prior to their injection for high-performance liquid chromatographic measurement of their conten...

Automated and Manual In-Line Solid-Phase Sample Preparation for High-Performance Liquid Chromatography Analysis of Bittering Substances in Worts and Beers

The feasibility of in-line solid-phase treatment of samples (5–50 μl) of stout, ale, and lager beers prior to their injection for high-performance liquid chromatographic measurement of their contents of iso-α-acids was established using manual column-switching. Samples were injected directly onto a short (1 cm) precolumn packed with octadecyl-silica and were purged free from interfering substances by sequential step elution with solvents of decreasing polarity. The analytes were then recovered from the precolumn by elution with the chromatographic mobile phase and transferred to an analytical column. Different sample pretreatments using sequential step elution were then developed using fully automated column switching equipment. The protocols used were shown to give accurate and precise measurements of iso-α-acids and accomplished satisfactory “cleanup” of the beer samples. Automated column-switching methodology was then developed to analyze simultaneously the iso-α-acids, α-acids, and β-acids in wort samples, using a precolumn and two analytical columns. Column switching allowed satisfactory analyses of wort samples to be performed in one-third of the time taken by conventional isocratic elution. An electrochemical detector was shown to be more than 50 times more sensitive for the measurement of α-acids and β-acids than an ultraviolet detector. The methods were applied to the measurement of iso-α-acids in beers bittered either with hops or pre-isomerized hop extracts. In addition, the course of α-acid isomerization was monitored in kettles boiled at atmospheric pressure.

A method for the measurement of a diene-conjugated derivative of linoleic acid, 18:2(9,11), in serum phospholipid, and possible origins.

A method is described for the measurement of a diene-conjugated derivative of linoleic acid, 18:2(9,11), in the phospholipid fraction of serum. The method is based on enzymatic hydrolysis, solid-phase sample preparation, and high-performance liquid chromatography (HPLC). Phospholipid esterified 18:2(9,11) can be detected in serum from normal individuals, and a provisional normal range is provided. The possible origins of 18:2(9,11) and the potential application of this method to the measurement of free radical damage to linoleic acid in vivo is discussed.

Analysis of vanillylmandelic and homovanillic acids in urine using solid phase sample preparation and high pressure liquid chromatography with electrochemical detection.

A rapid, simple and specific method for quantification of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in urine is described. The method is suitable for routine use and may include other organic acids in human urine.

Rapid high-performance liquid chromatographic method for the measurement of atenolol in plasma using UV detection

A rapid, selective and reproducible high-performance liquid chromatographic method has been developed for the measurement of the β-adrenoceptor blocking drug atenolol in small (400 μl) volumes of plasma. Following solid phase sample preparation using Bond-Elut TM mini-columns the compound is separated by high-performance column liquid chroamtography on a microparticulate (6 μm) cyano column using acetonitrile—ammonium dihydrogen phosphate (4:96) containing triethylamine (0.25%, v/v) as the mobile phase, and the absorption of the column effluent is monitored at 224 nm. The practical limit of quantitation, based upon an assay volume of 400 μl, is 25 ng/ml for atenolol. The average coefficient of variation is 3.1%.