Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine

A molecularly imprinted polymer, MIP, was prepared and evaluated as SPE sorbent for a cyclicized adduct formed to N-terminal valine (Pyr-Val) in hemoglobin from 1,2:3,4-diepoxybutane (DEB). This metabolite plays an important role in the carcinogenesis of 1,3-butadiene. The hydrazide of Pyr-Val, formed after hydrazinolysis of hemoglobin, as well as necessary standards was synthesized. The MIP was prepared from methacrylic acid with a structure analogue to the investigated adduct as template and the method was developed for aqueous conditions. Selective desorption was achieved when the sample was washed with water after loading in 10% acetonitrile. The primary interaction with the binding sites in the imprints was most likely of ionic character. Quantification of the Pyr-Val adduct was performed with LC/ESI-MS/MS, yielding an instrumental LOD of 150 pg injected amount.     

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means ± standard deviations: 0.68 ± 0.29 and 0.64 ± 0.28 nM, respectively), from pregnant women at term (1.37 ± 0.42 nM), and from umbilical vein (1.26 ± 0.33 nM) and umbilical artery (1.14 ± 0.35 nM), in milk (0.12 ± 0.05 nM) and from amniotic (0.03 ± 0.02 nM), peritoneal (0.93 ± 0.27 nM), follicular (1.17 ± 0.51 nM), and ovarian cyst (0.32 ± 0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n = 15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Quantitation of psilocin in human plasma by high-performance liquid chromatography and electrochemical detection: comparison of liquid–liquid extraction with automated on-line solid-phase extraction

Two modifications of the HPLC–ED method with respect to extraction procedure used have been developed for psilocin, the active metabolite of psilocybin, in human plasma using either liquid–liquid extraction (LLE) or automated on-line solid-phase extraction (on-line SPE). Each type of the sample preparation required a different HPLC system followed by electrochemical detection at 650 to 675 mV. The limit of quantitation of both modifications was 10 ng/ml psilocin. There was no significant difference observable between the LLE and the on-line SPE in terms of method standard deviation (LLE 1.82%, on-line SPE 1.13%) and the analytical results. However, the advantages of on-line SPE in addition to different selectivity were less manual effort, smaller plasma volumes of 400 μl (LLE 2 ml) and a recovery of psilocin in human plasma of nearly 100% (LLE 88%). In contrast to a previous procedure both methods were rapid, simple and reliable and yielded high plasma recoveries. They were used successfully in the quantitation of psilocin in plasma samples obtained from healthy volunteers after p.o. administration of 0.2 mg psilocybin per kg body mass. Plasma concentration curves and pharmacokinetic parameters were calculated.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Quantitation of trimipramine enantiomers in human serum by enantioselective high-performance liquid chromatography and mixed-mode disc solid-phase extraction

A sensitive and stereospecific method for the quantitation of trimipramine enantiomers in human serum was developed. The assay involves the use of a novel mixed-mode disc solid-phase extraction for serum sample clean-up prior to HPLC analysis and is also free of interference from the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine, the three major metabolites of trimipramine. Chromatographic resolution of trimipramine enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R) under isocratic conditions using a mobile phase consisting of 0.3 M aqueous sodium perchlorate-acetonitrile (58:42, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R- and S-trimipramine enantiomers were in the range of 93–96% at 25–185 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 0.30-8.00% and 1.60-10.20% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.01–2.10% and 1.00–3.00% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 15–250 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 15 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 10 ng/ml (S/N =2). In addition, separation of the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine were investigated. The desmethyltrimipramine enantiomers could be resolved on the Chiralcel OD-R column under the same chromatographic conditions as the trimipramine enantiomers, but the other two metabolite enantiomers required different mobile phases on the Chiralcel OD-R column to achieve satisfactory resolution with R_s values of 1.00.     

Application of molecularly imprinted polymers to solid-phase extraction of analytes from real samples

A review is presented of recent developments in the use of molecularly imprinted polymers (MIPs) as selective materials for solid-phase extraction. Compared with traditional sorbents, MIPs can not only concentrate but also selectively separate the target analytes from real samples, which is crucial for the quantitatively determination of analytes in complex samples. Consequently, as one of the most effective sorbents, MIPs have been successfully applied to the pretreatment of analytes in foods, drugs, and biological and environmental samples in the past five years.     

Determination of XR510, a balanced angiotensin II receptor antagonist, in dog and rat plasma by combined liquid-liquid/solid-phase extraction and high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the determination of XR510 (I), a new non-peptide angiotensin II (AII) receptor antagonist with balanced affinity for AT₁ and AT₂ receptor subtypes is described. I and the internal standard, XR513, were extracted from acidified plasma by combined liquid-liquid/solid-phase extraction and chromatographed on a phenyl column with ultraviolet absorbance detection at a wavelength of 272 nm. The mobile phase consisted of a mixture of acetonitrile and sodium phosphate buffer. For both rat and dog plasma, the limit of quantitation was 5 ng/ml. This method has been validated and successfully utilized to investigate the disposition of I.     

Suitability testing of commercial solid-phase extraction sorbents for sample clean-up in systematic toxicological analysis using liquid chromatography-(tandem) mass spectrometry

An entire series of SPE sorbents, classified into three different categories (apolar, mixed-mode and polymeric) was evaluated for sample preparation of a data-dependent LC-MS-MS "general unknown" screening procedure. An extraction procedure was formulated for each individual column, in agreement with the enclosed instructions, according to the characteristics of each packing. For conciseness, only neutral and basic compounds were chosen for this sorbent suitability test. Thus, the goal of our research was to select the best sorbent with regard to extraction yield and cleanliness of the extracts, all with respect to data-dependent acquisition (DDA) mediated LC-MS-MS general unknown screening. We conclude that for that purpose an Isolute C(8) sorbent performs best in terms of extraction yield and clean-up potential.     

Measurement of glycolysis reactants by high-throughput solid phase extraction with tandem mass spectrometry: Characterization of pyrophosphate-dependent phosphofructokinase as a case study

Glycolysis is a 10-step metabolic pathway involved in producing cellular energy. Many tumors exhibit accelerated glycolytic rates, and enzymes that participate in this pathway are focal points of cancer research. Here, a novel method for the measurement of glycolysis reactants from in vitro samples is presented. Fast and direct measurement is achieved by an automated system that couples on-line solid phase extraction (SPE) with tandem mass spectrometry (MS/MS). The single analytical method enables multiple reactants to be measured concurrently, sustains a cycle time of 8 s, and permits the measurement of up to 10,000 samples per day. Concentration–response curves were conducted using standards for 10 metabolic intermediates, and the results demonstrate that the detection strategy has excellent sensitivity (average limit of detection = 5.4 nM), dynamic range (nanomolar to micromolar), and linear response (average R² = 0.998). To test the analysis method on reactions, pyrophosphate-dependent phosphofructokinase (PPi–PFK) was used as a model system. Data that corroborate the activation and inhibition of PPi–PFK are presented, and the ways in which SPE–MS/MS simplifies experimental design and interpretation are highlighted. In summary, the method for measuring metabolic intermediates described here demonstrates unprecedented speed, performance, and versatility.     

Detection of the acrolein-derived cyclic DNA adduct by a quantitative 32P-postlabeling/solid-phase extraction/HPLC method: blocking its artifact formation with glutathione

Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N2-propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1 fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3'-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship.     

Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250 ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500 ng/mL for HHMA, and 150–7500 ng/mL for HHA (r² > 0.993 for all analytes). The lower limits of quantification were 12.5 ng/mL urine for MDMA, MDA, HMA and HMMA, 25 ng/mL for HHMA and 150 ng/mL for HHA. Reproducibility was good (intra-assay precision = 1.7–6.1%; inter-assay precision = 0.6–5.7%), as was accuracy (intra-assay deviation = 0.1–4.8%; inter-assay deviation = 0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV < 8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24 h from rats given subcutaneous MDMA.     

Anandamide levels in human female reproductive tissues: Solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n = 14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5 pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 ± 1.04 pmol/g for placenta and 1.19 ± 0.68 pmol/g for fetal membranes, and 0.93 ± 0.28, 0.88 ± 0.33, 0.77 ± 0.30, and 0.06 ± 0.04 nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P < 0.0001), in umbilical vein compared with umbilical artery (P = 0.0015), and in plasma from maternal circulation compared with umbilical artery (P = 0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.     

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection

In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.     

Current trends in solid-phase-based extraction techniques for the determination of pesticides in food and environment

Solid-phase extraction (SPE) procedures for pesticide residues in food and environment are reviewed and discussed. The use of these procedures, which include several approaches such as: matrix solid-phase dispersion (MSPD), solid-phase micro-extraction (SPME) and stir-bar sorptive extraction (SBSE), represents an opportunity to reduce analysis time, solvent consumption, and overall cost. SPE techniques differ from solvent extraction depending on the interactions between a sorbent and the pesticide. This interaction may be specific for a particular pesticide, as in the interaction with an immunosorbent, or non-specific, as in the way a number of different pesticides are adsorbed on apolar or polar materials. A variety of applications were classified according to the method applied: conventional SPE, SPME, hollow-fiber micro-extraction (HFME), MSPD and SBSE. Emphasis is placed on the multiresidue analysis of liquid and solid samples.     

On-line extraction using an alkyl-diol silica precolumn for racemic citalopram and its metabolites in plasma: Results compared with solid-phase extraction methodology

Sample preparation is usually the most critical and time consuming step when using HPLC for drug analysis in biological matrixes. Sample extracts have to be clean considering both chromatographic interferences and column maintenance. To meet some of these criteria a fully automated on-line extraction (OLE) analysis method was developed for the antidepressant drug citalopram and its two demethylated metabolites, using an RP-C4-ADS extraction column. A comparison between the new OLE method and an off-line solid-phase extraction method showed that the two methodologies were equal in analytical precision but that the OLE method was faster and therefore superior in sample capacity per day.     

Development of a novel method for triterpenoidal saponins in rat plasma by solid-phase extraction and high-performance liquid chromatography tandem mass spectrometry

A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3, and 23-hydroxybetulinic acid) in rat plasma following solid-phase extraction (SPE). The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multiswitching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18 column (250 × 4.6 mm, 5 μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as follows: m/z 1219.7/749.4 for anemoside B4, m/z 819.4/347.2 for pulsatilloside B, m/z 749.6/471.2 for anemoside A3, m/z 471.4/471.4 for 23-hydroxybetulinic acid, and m/z 461.1/285.0 for the internal standard. The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.     

Evaluation of online extraction/mass spectrometry for in vivo cassette analysis

An online extraction/mass spectrometry technique was evaluated for direct analysis of plasma samples. A simple user-friendly online extraction system that consists of two pumps, an autosampler, a six-port switching valve and a mass spectrometer is described. The system was controlled by the LC-MS software (Masslynx 3.5, Waters Corporation, Beverly, MA). Various analytical conditions such as extraction column, mobile phases, run time and wash solvent were optimized to establish an analytical method that was simple, easy to set up and generic. Sample preparation effort was minimal, which included dilution of plasma with water and centrifugation conducted in 96-well plate format. The system was used to analyze in vivo plasma samples from rat n-in-one cassette dosing studies. Concentration and pharmacokinetic (PK) data obtained from the online extraction method were comparable with data obtained from the protein precipitation extraction method. Overall, the simple, robust online extraction system provides cost savings by minimizing sample preparation and method development time. The system was used to analyze compounds from different structural classes. These studies suggest that calculated lipophilicity of a compound can be used as a tool for pre-selection of extraction column, which would save method development time for early discovery studies.     

Feasibility of the direct coupling of solid-phase extraction-pipette tips with a programmed-temperature vaporiser for gas chromatographic analysis of drugs in plasma

Solid-phase extraction-pipette tips (SPE-PTs) were used for micro solid-phase extraction of lidocaine and diazepam from plasma. Off-line extraction was followed by on-line desorption. On-line desorption was carried out by direct coupling of the SPE-PTs with the liner of the programmed-temperature vaporiser. This coupling only required shortening of the liner by maximally 16 mm, cutting the SPE-PT, and equipping the remaining part with two O-rings. Due to the heating of the injector the SPE-PTs were heated as well, which resulted in a significant amount of impurities. Pre-heating and pre-washing was performed prior to the extraction to reduce the impurity level. The internal coupling device was applied successfully for the analysis of plasma samples with gas chromatography (GC) and mass-selective detection. Detection limits of 0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam, respectively, using 200 microl plasma. Recoveries for both compounds were about 80%. Although it is possible, the internal coupling device was not developed to be used as such. The main goal of this coupling was to show the feasibility of the integration of SPE-PTs with GC and to realize an important step to new automated SPE-GC systems.     

Determination of tripdiolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reversed-phase high-performance liquid chromatography

Extracts of Tripterygium wilfordii Hook F. have been widely used in China to treat a variety of autoimmune and inflammatory diseases. The diterpenoids triptolide and tripdiolide are two major active components in the T. wilfordii ethyl acetate extract. An efficient solid-phase extraction and high-performance liquid chromatography (SPE-HPLC) method to measure triptolide content in the extract has been previously reported. However, a suitable means of tripdiolide quantification is not available because of interfering compounds in the extract that co-elute with tripdiolide. Therefore, this paper describes a method wherein tripdiolide content can be measured from a small amount of the extract. The extract solution (600 microL) was applied into an aminopropyl SPE tube. Triptolide was eluted with dichloromethane:methanol (1 mL, 49:1 v/v), followed by tripdiolide elution with dichloromethane:methanol (3 mL, 17:3 v/v). The tripdiolide eluate was analysed by HPLC using an isocratic solvent system and was quantified by measuring the peak area at 219 nm. The contents of triptolide and tripdiolide in the extract were determined to be 807.32 +/- 51.94 and 366.13 +/- 17.21 microg/g of extract, respectively. Since tripdiolide is biologically active and makes up a considerable portion of the extract, for extract quality control and standardisation purposes, it should be measured along with triptolide using the proposed SPE-HPLC method.