Solid-phase Extraction Enrichment Research Articles

Nitro Indole Derivatives as Novel Dual-Polarity Matrices for MALDI Mass Spectrometry and Imaging with Broad Applications.

A new matrix framework is presented in this study for the improved ionization efficiency of complex mixtures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry/imaging. Five nitro indole (NI) derivatives [3-methyl-4-nitro-1H-indole (3,4-MNI), 3-methyl-6-nitro-1H-indole (3,6-MNI), 2,3-dimethyl-4-nitro-1H-indole (2,3,4-DMNI), 2,3-dimethyl-6-nitro-1H-indole (2,3,6-DMNI), and 4-nitro-1H-indole (4-NI)] were synthesized and shown to produce both positive and negative ions with a broad class of analytes as MALDI matrices. NI matrices were compared to several common matrices, such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxylcinnamic acid (CHCA), sinapinic acid (SA), 1,5-diaminonaphthelene (1,5-DAN), and 9-aminoacridine (9-AA), for the analysis of lipid, peptide, protein, glycan, and perfluorooctanesulfonic acid (PFOS) compounds. 3,4-MNI demonstrated the best performance among the NI matrices. This matrix resulted in reduced ion suppression and better detection sensitivity for complex mixtures, for example, egg lipids/milk proteins/PFOS in tap water, while 2,3,6-DMNI was the best matrix for blueberry tissue imaging. Several important aspects of this work are reported: (1) dual-polarity ion production with NI matrices and complex mixtures; (2) quantitative analysis of PFOS with a LOQ of 0.5 ppb in tap water and 0.05 ppb in MQ water (without solid phase extraction enrichment), with accuracy and precision within 5%; (3) MALDI imaging with 2,3,6-DMNI as a matrix for plant metabolite/lipid identification with ionization enhancement in the negative ion mode m/z 600-900 region; and (4) development of a thin film deposition under/above tissue method for MALDI imaging with a vacuum sublimation matrix on a high-vacuum MALDI instrument.

An Alternative Strategy for Screening and Confirmation of 330 Pesticides in Ground- and Surface Water Using Liquid Chromatography Tandem Mass Spectrometry.

The presence of pesticide residues in water is a huge worldwide concern. In this paper we described the development and validation of a new liquid chromatography tandem mass spectrometric (LC-MS/MS) method for both screening and quantification of pesticides in water samples. In the sample preparation stage, the samples were buffered to pH 7.0 and pre-concentrated on polymeric-based cartridges via solid-phase extraction (SPE). Highly sensitive detection was carried out with mobile phases containing only 5 mM ammonium formate (pH of 6.8) as an eluent additive and using only positive ionization mode in MS/MS instrument. Hence, only 200-fold sample enrichment was required to set a screening detection limit (SDL) and reporting limit (RL) of 10 ng/L. The confirmatory method was validated at 10 and 100 ng/L spiking levels. The apparent recoveries obtained from the matrix-matched calibration (5–500 ng/L) were within the acceptable range (60–120%), also the precision (relative standard deviation, RSD) was not higher than 20%. During the development, 480 pesticides were tested and 330 compounds fulfilled the requirements of validation. The method was successfully applied to proficiency test samples to evaluate its accuracy. Moreover, the method robustness test was carried out using higher sample volume (500 mL) followed by automated SPE enrichment. Finally, the method was used to analyze 20 real samples, in which some compounds were detected around 10 ng/L, but never exceeded the assay maximum level.

Simultaneous determination of seven bisphenol analogues in surface water by solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry

For the simultaneous determination of seven types of bisphenol analogues (BPs) in surface water, a method combining solid-phase extraction (SPE) with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed. The extraction efficiency was improved by optimizing the parameters that affect it, including the sorbent types of SPE cartridge, pH, enrichment factor, flow rate, rinsing and elution volume. On the basis of the single factor experiments, an attempt to obtain the result of optimizing by response surface methodology was made, and the optimal parameters were as follows: at a flow rate of 8 mL/min, 1000 mL of water sample was run through the hydrophilic-lipophilic balance cartridge, rinsed with 10% methanol, eluted with 9 mL methanol, and concentrated with nitrogen using an automatic SPE system. The analytes were separated on an Acquity UPLC BEH C18 column using gradient elution with a mobile phase of acetonitrile and 0.05% (v/v) ammonia, and then analyzed using UPLC-MS/MS with multiple reaction monitoring in the negative-ion mode, with an 8-minute chromatographic run time. In the range of 1.0–500 μg/L, seven BPs showed good linearity with the correlation coefficient greater than 0.998. The recovery ranged from 82.3 to 96.7%, and the range of matrix effect was 89.2 to 107%. These results indicated excellent recovery and minimal matrix effect. The limits of detection were 0.05–1.5 ng/L, the relative standard deviation ranged from 2.9 to 8.5%. Applying the developed method, seven BPs in Luoma Lake and its inflow rivers were analyzed. Only five BPs were detected in Luoma Lake and four BPs were detected in inflow rivers, and the concentrations of the detected BPs were determined. BPA and BPF were the predominant BPs, and the concentration of BPA was the highest.

Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45-50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100-1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.

Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

Analysis, Formation and Inhibition of Cholesterol Oxidation Products in Foods: An Overview (Part I)

Cholesterol oxidation products (COPs) formed in cholesterol-containing foods during heating or illumination have been found to impart a potential hazard to health. Numerous studies have indicated that COPs may have several adverse biological effects, such as mutagenicity, carcinogenicity, angiotoxicity, cytotoxicity, atherogenicity, atherosclerosis, cell membrane damage and inhibition of cholesterol biosynthesis. Therefore, the safety of COPs has become a major concern for the public. This paper is an overview of analysis, formation and inhibition of COPs in foods. COPs are routinely extracted by organic solvents, followed by saponification and solid phase extraction for enrichment of COPs, and separation and identification by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). The identification and quantification of COPs using the GC-MS technique was found to be rapid and sensitive, however, the formation of artifacts is a major drawback. The HPLC method failed to resolve several geometrical isomers, and double bond-free COPs such as isomeric 5,6-epoxides and triol could not be detected with UV. The oxidation of cholesterol can be accelerated by heating, pH, storage conditions, the presence of food components and other factors. Several COPs are commonly present in food systems, including 7α-OH, 7β-OH, 5,6α-EP, 5,6β-EP, 7-keto, 20α-OH, 25-OH and triol. Of these COPs, 5,6α-EP, 5,6β-EP, 7-keto, 20α-OH and 25-OH are primary oxidation products, while 7α-OH, 7β-OH and triol are secondary products. Some antioxidants have been found to reduce the formation of COPs in an appropriate concentration. Also, adequate packaging is necessary to provide a physical barrier for air and light, and thus minimize cholesterol oxidation. Further research is necessary to study how to inhibit COPs formation in foods.

In situ growth of metal-organic framework HKUST-1 in an organic polymer as sorbent for nitrated and oxygenated polycyclic aromatic hydrocarbon in environmental water samples prior to quantitation by HPLC-UV.

An in situ preparation of metal-organic framework (HKUST-1) on the surface of an organic polymer is reported. The hybrid material was evaluated as sorbent for solid-phase extraction (SPE) of oxygenated and nitrated polycyclic aromatic hydrocarbons (PAHs). The growth of HKUST-1 on MAA-based polymer was accomplished using a layer-by-layer assembly strategy. The HKUST-1-polymer was characterized using powder X-ray diffraction and scanning electron microscopy to demonstrate the incorporation of MOF crystals onto the polymer surface. The MOF (nano)crystals were characterized using high-resolution transmission electron microscopy and high-angle annular-dark-field scanning transmission electron microscopy. The water-stable MOF-polymer was evaluated as SPE sorbent, and several variables that can influence the extraction recoveries of PAH derivatives were investigated. Under the selected conditions, the detection limits varied between 4 and 21ng·L-1, and the precision (relative standard deviation) was below 12%. The recovery values for spiked solutionsranged from 95 to 104%, and the enrichment factor achieved was close to 1000-fold using a high sample volume (100mL) and very low desorption volume (100μL). The SPE enrichment combined with HPLC and DAD detector was successfully applied to the extraction and determination of polycyclic hydrocarbons in environmental water samples. Graphical abstract A layer-by-layer synthesis of HKUST-1 onto polymer surface was performed and applied to isolation of PAH derivatives in environmental water samples.

Selective extraction and enrichment of aflatoxins from food samples by mesoporous silica FDU-12 supported aflatoxins imprinted polymers based on surface molecularly imprinting technique

In this work, a highly selective surface molecular imprinted polymers (FDU-12@MIPs) were prepared using structural analogues of aflatoxins as the template molecule and mesoporous silica FDU-12 as the carrier. The resulting polymers were characterized by scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction and attenuated total reflection Fourier transform infrared spectrometer. A highly ordered mesoporous nanostructure was observed in the fabricated polymer. Results showed that the adsorption capacity of FDU-12@MIPs for aflatoxins were higher than that of non-imprinted mesoporous silica polymer (FDU-12@NIPs). FDU-12@MIPs were successfully used as a solid-phase extraction sorbent coupled with HPLC for aflatoxin G2, G1, B2 and B1 determination in food samples. Under optimized conditions, recoveries from the spiked cereals were found to be 82.6%–116.7%, with relative standard deviations ranging from 2.73 to 4.21. The detection limits of aflatoxin G2, G1, B2 and B1 were estimated to be 0.05 μg kg−1, 0.06 μg kg−1, 0.06 μg kg−1 and 0.05 μg kg−1, respectively. Limits of quantifications were estimated to be 0.15 μg kg−1, 0.2 μg kg−1, 0.2 μg kg−1and 0.15 μg kg−1, respectively. The developed method exhibited a favorable linear response toward aflatoxin G2, G1, B2 and B1 in the range of 0.1 μg kg−1-50 μg kg−1, R2 is 0.9992–0.9996. These results indicated that FDU-12@MIPs could be used as an efficient adsorbent of solid phase extraction for enrichment of aflatoxins in real samples.

Solid-phase extraction-stepwise elution (SPE-SE) procedure for isolation of dissolved organic matter prior to ESI-FT-ICR-MS analysis

Characterization of dissolved organic matter (DOM) at the molecular level will greatly improve our understanding of its bio-geochemical role in controlling the fate of contaminants in the environment, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is the most powerful analytical technique for this purpose. Before FT-ICR-MS analysis, isolation, desalination and concentration of DOM are necessary, and solid-phase extraction (SPE) is the most widely applied pretreatment procedure. However, some molecular information is lost using conventional SPE methods. Here, we propose a novel strategy of SPE enrichment using stepwise elution (SPE-SE). Compounds in DOM were divided into three fractions by this SPE-SE procedure according to their polarity and ionization efficiency. The diversity of DOM molecules identified by ESI-FT-ICR-MS using SPE-SE exceeded those using conventional SPE methods by more than 50%. This method is feasible and has the potential to be used as a pretreatment strategy for complex DOM matrixes prior to ESI-FT-ICR-MS analysis, especially for those rich in nitrogenous molecules, carbohydrates, lipids and/or aromatic compounds.

Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti4+-SPE enrichment for mass spectrometric analysis

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti4+-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti4+-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.

A novel mass spectrometry‐based method for determining insulin‐like growth factor 1: assessment in a cohort of subjects with newly diagnosed acromegaly

To develop an alternative method to immunoassay for the quantitative analysis of insulin-like growth factor 1 (IGF-1) using a mass spectrometry (MS)-based approach. A stable isotope dilution Ultra High Performance Liquid Chromatography tandem MS (uHPLC-MS/MS)-based method for the quantification of IGF-1 was developed. The method employed Selected Reaction Monitoring (SRM) of two tryptic peptides derived from IGF-1, and utilised solid phase extraction for enrichment of the peptide fraction containing IGF-1 rather than immunocapture, so was less susceptible to assay interference. Plasma samples from 25 consecutive unselected patients with newly diagnosed acromegaly, collected both before and after 24 weeks of primary medical therapy with Lanreotide Autogel(®), were analysed by a widely used commercial immunoassay (Siemens Immulite 2000(®)) and by uHPLC-MS/MS. The uHPLC-MS/MS method showed good correlation with the immunoassay over a wide range of IGF-1 concentrations. The Passing and Bablock regression was: uHPLC-MS/MS (nmol/l) = 1.37 (95% confidence interval: 1.26-1.46) × immunoassay (nmol/l) + 3.14 (95% confidence interval: -2.71 to 10.32). Six patients had discordant growth hormone (GH) and IGF-1 levels following primary medical therapy, and in all six the immunoassay and uHPLC-MS/MS platforms returned comparable results. The method was not affected by concentrations of IGFBP3 up to 12,500 ng/ml. uHPLC-MS/MS offers an independent method for determining/validating IGF-1 in subjects with acromegaly. Further studies, including the establishment of age- and sex-matched reference ranges and calibration to the new International IGF-1 standard IS 02/254, are now required to allow its introduction in to routine clinical use.

Determination of Sulfonamides in Milk Samples Based on RAFT Molecularly Imprinted Solid Phase Extraction

Abstract. A novel molecularly imprinted polymer (MIP) with high selectivity for sulfonamides was synthesized using reverisible addition fragmentation chain transfer (RAFT) polymerization technique with 2-cyanoprop-2-yl-dithionaphthalate as RAFT reagent, sulfadimethoxine as template, methacrylic acid (MAA) as functional monomer. The obtained MIP was characterized by static adsorption and the results indicated that the MIPs had high adsorption affinity to sulfonamides. The MIP was sucessfully empolyed as the adsorbtion sorbent of solid phase extraction for enrichment and clean-up four sulfonamides from food samples. Under the optimized conditions, good linearities were ranged from 0.010-10 µg/mL with r2≥0.9991.The recoveries at three spiked levels were ranged from 91.0-110.2% with RSD less than 5.8%.

Mercury determination and speciation analysis in surface waters

Abstract A sorbent L-cysteine grafted silica gel has been evaluated for separation and enrichment of dissolved inorganic i-Hg(II) and methylmercury CH3Hg(I) from surface waters at sub-µg L−1 concentrations. Chemical parameters for mercury species enrichment and separation have been optimized. Analytical schemes for the determination of Hg species, using selective column solid phase extraction (SPE) with continuous flow chemical vapor generation atomic absorption spectrometry (CF-CVG-AAS) or inductively coupled plasma-mass spectrometry (ICP-MS) were developed. Possibilities for on-site SPE enrichment were demonstrated as well. The limits of quantification were 1.5 and 5 ng L−1 for dissolved i-Hg(II) and CH3Hg(I) by CF-CVG-AAS and 1 and 2.5 ng L−1 by ICP-MS with relative standard deviations between 7–12% and 7–14%, respectively. The chemically modified SPE sorbent has demonstrated high regeneration ability, chemical and mechanical stability, acceptable capacity and good enrichment factors. Results for total dissolved mercury were in reasonable agreement with those from independent analyses by direct ICP-MS determinations for river waters and for estuarine water certified reference material.

Investigation of acidic pharmaceuticals in river water and sediment by microwave-assisted extraction and gas chromatography–mass spectrometry

Investigations were performed along the Danube river at Budapest (Hungary) by collecting water and sediment samples simultaneously for 1 year in order to clarify the possible hazard of selected acidic pharmaceuticals (ibuprofen, naproxen, ketoprofen, and diclofenac) on the water supply used for the production of drinking water by bank filtration. In the case of water samples, the sample preparation procedure included solid phase extraction (SPE), meanwhile, in the case of sediment samples, microwave-assisted extraction (MAE) followed by dispersive matrix extraction (DME) for pre-cleaning as well as SPE for enrichment. The quantification was carried out using gas chromatography–mass spectrometry (GC–MS). The calculated recoveries were 97–99% (± 7%) for the water and 95–103% (± 12%) for the sediment samples. In the river water, ketoprofen concentration was always below the limit of quantification (LOQ) level; ibuprofen, naproxen and diclofenac could be quantified in the range of 8–50, 2–30, 7–90 ng/L. In sediments, only naproxen and diclofenac were found in the range of 2–20 and 5–38 ng/g, respectively. According to the obtained results, the concentration ratios of the two phases linearly depended on the total organic carbon content (TOC) of the sediments at each sampling date. The linear regressions were 0.925 and 0.946 for naproxen and diclofenac, respectively.

A sensitive and high-throughput LC–MS/MS method for the quantification of pegylated-interferon-α 2a in human serum using monolithic C 18 solid phase extraction for enrichment

The analysis of pegylated-interferon-α 2a in patient serum samples is of high interest for clinic research trials, as this therapeutic protein has become an important antiviral treatment. In this study, an LC–MS/MS method for the absolute quantification of pegylated-interferon-α 2a in human serum was developed. The assay achieved a lower limit of quantification of 3.6 ng/mL (60 pM) with the use of a monolithic C 18 solid phase extraction to enrich the target protein. The linear range of the assay was defined up to 54 ng/mL to measure the typical clinical pegylated-interferon-α 2a levels, and within this range, the precision and accuracy were found to be within ±20%. The method was applied to a clinical study and found suitable for high-throughput analysis of pegylated-interferon-α 2a in human serum. In addition, further investigations suggested the enrichment step may have general application to the sensitive analysis of other low molecular weight proteins.

2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C18 “transition” SPE columns. A PLRP C18 column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.

Combination of supported liquid membrane and solid-phase extraction for sample pretreatment of triazine herbicides in juice prior to capillary electrophoresis determination

A possibility of a combination of supported liquid membrane (SLM) and solid-phase extraction (SPE) for the determination of atrazine at microgram level in different type of fruit juices is presented. In comparison to SPE extraction from juice samples, the application of SLM–SPE enrichment provides much cleaner extracts and the possibility of lowering the limit of detection as low as 30 μg/l. However, it was also shown that by appropriate manipulation of SLM extraction conditions mainly flow-rate of donor phase and volume ratio between donor and acceptor phase, the level of detection can be further decreased to 10 μg/l. The results suggest that the application of SLM extraction prior to SPE is an alternative method for atrazine enrichment from complicated liquid matrices and could be used as routine method for the clean-up of such samples.

Determination of angiotensin-converting enzyme inhibitory peptide Leu-Lys-Pro-Asn-Met (LKPNM) in bonito muscle hydrolysates by LC-MS/MS.

Proteolytic digestion of dried bonito muscle with thermolysin produces a hydrolysate with strong angiotensin-converting enzyme (ACE) inhibitory activity and is the basis of a dietary supplement with antihypertensive activity. A major portion of the ACE activity was shown previously to arise from the peptide Leu-Lys-Pro-Asn-Met (LKPNM). A straightforward method to quantify this peptide was developed using one-step C18 solid-phase extraction (SPE) followed by LC-MS/MS quantification. The SPE step resulted in a hydrolysate that was still crude, as illustrated by combined size-exclusion chromatography/multi-angle laser light scattering detection that showed that a major fraction of oligopeptides were in the 2-20 kDa range. This fraction has a weight-average molecular weight (M(w)) of approximately 5.0 kDa. Method validation for specificity, linearity, accuracy, precision, and reproducibility showed that standard additions of synthetic LKPNM to bonito extract with SPE enrichment followed by LC-MS/MS is a suitably robust procedure for the determination of LKPNM content. The method was also successful for encapsulated powders in which the excipients used are insoluble in water and could be removed by centrifugation.

Analytical methods for the determination of pharmaceuticals in aqueous environmental samples

Recently several methods have been developed for the determination of drugs and their metabolites in the lower ng/l range using solid phase extraction (SPE), derivatization, detection and confirmation by gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS or LC–electrospray tandem MS (LC–ES/MS/MS). A wide range of pharmaceuticals from different medicinal classes can be determined down to the lower ng/l range. Due to the basically elevated polarity of pharmaceuticals either analysis by LC–ES/MS/MS or an efficient derivatization prior to measurements by GC/MS are mostly essential. A direct comparison of GC/MS and LC–ES/MS/MS displayed that only the latter allows for the analysis of the extreme polar betablockers atenolol and sotalol due to an incomplete derivatization of the functional groups. Further, the relative standard deviation using LC–ES/MS/MS was lower. However, when analyzing highly contaminated samples such as sewage a suppression of the electrospray ionization is likely to occur. Thus, to guarantee accurate and reproducible data either an efficient clean-up step has to be included into the sample preparation or an appropriate surrogate standard has to be spiked prior to SPE enrichment.

Solid phase extraction enrichment of pesticide mixtures from aqueous solutions: a quantitative FT‐IR analysis

Solid phase extraction of ten commonly used pesticides on activated carbon has been investigated. Enrichment of these pesticides was successfully achieved at ppm levels and samples were then analysed using FT‐IR. The enrichment technique was employed for each pesticide sample individually and their complex mixtures. The FT‐IR technique was employed for the quantitative analysis. According to the results obtained from FT‐IR some pesticides were recovered at 36.8–86.5% whereas 2,4‐D and endosulfan were decomposed during the extraction procedure.