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The global livestock sector, particularly ruminants, contributes substantially to the total anthropogenic greenhouse gases. Management and dietary solutions to reduce enteric methane (CH 4 ) emissions are extensively researched. Animal breeding that exploits natural variation in CH 4 emissions is an additional mitigation solution that is cost-effective, permanent, and cumulative. We quantified the effect of including CH 4 production in the Dutch breeding goal using selection index theory. The current Dutch national index contains 15 traits, related to milk yield, longevity, health, fertility, conformation and feed efficiency. From the literature, we obtained a heritability of 0.21 for enteric CH 4 production, and genetic correlations of 0.4 with milk lactose, protein, fat and DM intake. Correlations between enteric CH 4 production and other traits in the breeding goal were set to zero. When including CH 4 production in the current breeding goal with a zero economic value, CH 4 production increases each year by 1.5 g/d as a correlated response. When extrapolating this, the average daily CH 4 production of 392 g/d in 2018 will increase to 442 g/d in 2050 (+13%). However, expressing the CH 4 production as CH 4 intensity in the same period shows a reduction of 13%. By putting economic weight on CH 4 production in the breeding goal, selective breeding can reduce the CH 4 intensity even by 24% in 2050. This shows that breeding is a valuable contribution to the whole set of mitigation strategies that could be applied in order to achieve the goals for 2050 set by the EU. If the decision is made to implement animal breeding strategies to reduce enteric CH 4 production, and to achieve the expected breeding impact, there needs to be a sufficient reliability of prediction. The only way to achieve that is to have enough animals phenotyped and genotyped. The power calculations offer insights into the difficulties that will be faced in trying to record enough data. Recording CH 4 data on 100 farms (with on average 150 cows each) for at least 2 years is required to achieve the desired reliability of 0.40 for the genomic prediction., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The study aimed at developing a deep-learning (DL)-based algorithm to predict the virtual soft tissue profile after mandibular advancement surgery, and to compare its accuracy with the mass tensor model (MTM). Subjects who underwent mandibular advancement surgery were enrolled and divided into a training group and a test group. The DL model was trained using 3D photographs and CBCT data based on surgically achieved mandibular displacements (training group). Soft tissue simulations generated by DL and MTM based on the actual surgical jaw movements (test group) were compared with soft-tissue profiles on postoperative 3D photographs using distance mapping in terms of mean absolute error in the lower face, lower lip, and chin regions. 133 subjects were included - 119 in the training group and 14 in the test group. The mean absolute error for DL-based simulations of the lower face region was 1.0 ± 0.6 mm and was significantly lower (p = 0.02) compared with MTM-based simulations (1.5 ± 0.5 mm). CONCLUSION: The DL-based algorithm can predict 3D soft tissue profiles following mandibular advancement surgery. With a clinically acceptable mean absolute error. Therefore, it seems to be a relevant option for soft tissue prediction in orthognathic surgery. Therefore, it seems to be a relevant options., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Extracellular lipase from Yarrowia lipolytica was immobilized by ionotropic gelation with alginate and chitosan as encapsulating agents. Photomicrographs revealed a collapsed and heterogeneous surface of these microcapsules due to freeze-drying process. The optimum reaction temperature for the microencapsulated lipase (40 °C) was higher than for free lipase (35 °C) as well as the optimum pH (8.0 and 7.5, respectively). The study of the reaction kinetics showed that a higher maximum reaction rate (V max ) (221.1 U/mg) for the free lipase in comparison to the immobilized form (175.3 U/mg). A protective effect of the microcapsule was detected in the storage of the enzyme at room temperature, as after 75 days 35% of activity was maintained for the microcapsules, while no activity remained after 15 days with the free enzyme. Lower values for inactivation constant (k d ) and increase in half-life for immobilized lipase showed that lipase microencapsulation favored the thermostability of this enzyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Capillary electrophoresis (CE) played an important role in developments in the life sciences. The technique is nowadays used for the analysis of both large and small molecules in applications where it performs better than or is complementary to liquid chromatographic techniques. In this review, principles of different electromigration techniques, especially capillary isoelectric focusing (CIEF), capillary gel (CGE) and capillary zone electrophoresis (CZE), are described and recent developments in instrumentation, with an emphasis on mass spectrometry (MS) coupling and microchip CE, are discussed. The role of CE in the life sciences is shown with applications in which it had a high impact over the past few decades. In this context, current practice for the characterization of biopharmaceuticals (therapeutic proteins) is shown with CIEF, CGE and CZE using different detection techniques, including MS. Subsequently, the application of CGE and CZE, in combination with laser induced fluorescence detection and CZE-MS are demonstrated for the analysis of protein-released glycans in the characterization of biopharmaceuticals and glycan biomarker discovery in biological samples. Special attention is paid to developments in capillary coatings and derivatization strategies for glycans. Finally, routine CE analysis in clinical chemistry and latest developments in metabolomics approaches for the profiling of small molecules in biological samples are discussed. The large number of CE applications published for these topics in recent years clearly demonstrates the established role of CE in life sciences., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Radiation-free 3D post-operative sequential follow-up in craniosynostosis is hindered by the lack of consistent markers restricting evaluation to subjective comparison. However, using the computed cranial focal point (CCFP), it is possible to perform correct sequential image superposition and objective evaluation. We used this technique for mean volume and shape change evaluation of the head utilizing 3D photos after endoscopically assisted trigonocephaly surgery., Methods: We performed a mean head shape and volume evaluation on age grouped 3D photos (n = 86) of children who underwent endoscopically assisted strip craniectomy with helmet therapy. We used CT-scans of healthy children as reference. We performed a mean shape evolution analysis and calculated the anterior fossa to total volume ratio (A/T-ratio). The volume- and A/T-ratio pattern were compared with the reference group., Results: The mean anterior fossa volume evolved from 336 ml (33.4% A/T-ratio) pre-surgery to 664 ml (36.0% A/T-ratio) at 37-48 months post-surgery. Both groups have a near similar volume- and A/T-ratio pattern over time. The first 18 months show a predominant growth around the resected metopic suture. Between 18 and 24 months we observed mostly anterior orbital rim growth. From 24 months till 36-48 months the head grows predominantly at the temporal area. The least outward growth was observed at the temporal bones., Conclusion: Using a novel technique we were able to objectively evaluate head shape and volume using stereophotogrammetry after endoscopically assisted strip craniectomy. The A/T-ratio and volume growth pattern of endoscopically treated patients is near identical to that of the normal reference group., (Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Survival during the first year after first calving was investigated over the last 25 years, 1989-2013, as well as how the association of survival with season of calving, age at first calving (AFC) and within-herd production level has changed over that period. The data set contained 1 108 745 Dutch black-and-white cows in 2185 herds. Linear models were used to estimate (1) effect of year and season and their interaction and (2) effect of AFC, within-herd production level, and 5-year intervals and their two-way interactions, and the genetic trend. All models contained AFC and percentage of Holstein Friesian as a fixed effect, and herd-year-season, sire and maternal grandsire as random effects. Survival and functional survival were analysed. Functional survival was defined as survival adjusted for within-herd production level. Survival rate increased by 8% up to 92% in the last 25 years. When accounting for pedigree, survival showed no improvement up to 1999, but improved since then. Genetically, survival increased 3% to 4% but functional survival did not increase over the 25 years. We found an interesting difference between the genetic trends for survival and functional survival for bulls born between 1985 and 1999, where the trend for survival was still increasing, but was negative for functional survival. Since 1999, genetic trend picked up again for both survival and functional survival. AFC, season of calving and within-herd production level affected survival. Survival rate decreased 0.6%/month for survival and 1.5% for functional survival between AFC of 24 and 32 months. Calving in summer resulted in 2.0% higher survival than calving in winter. Within herd, low-producing cows had a lower survival rate than high-producing cows. However, these effects became less important during the recent years. Based on survival optimum AFC is around 24 months, but based on functional survival it is better to have an AFC<24 months. Overall, survival rate of heifers has improved considerably in the past 25 years, initially due to the focus on a high milk production. More recently, the importance of a high milk production has been reduced towards attention for functional survival.

The current methodology to identify allergenic food proteins is effective in identifying those that are likely to cross-react with known allergens. However, most assays show false positive results for low/non-allergens. Therefore, an ex vivo/in vitro DC-T cell assay and an in vivo mouse model were used to distinguish known allergenic food proteins (Ara h 1, β-Lactoglobulin, Pan b 1, bovine serum albumin, whey protein isolate) from low/non allergenic food proteins (soy lipoxygenase, gelatin, beef tropomyosin, rubisco, Sola t 1). CD4+ T cells from protein/alum-immunized mice were incubated with corresponding protein-pulsed bone marrow-derived DC and analyzed for cytokine release. All known allergens induced Th2 responses in vitro, whereas soy lipoxygenase, gelatin or beef tropomyosin did not. Sola t 1 and rubisco induced a more generalized T cell response due to endotoxin contamination, indicating the endotoxin-sensitivity of the DC-T assay. To analyze responses in vivo, mice were orally sensitized on days 0 and 7. Known allergens induced IgE and mMCP-1 release upon oral challenge at day 16, whereas the low/non-allergens did not. Both the DC-T cell assay and the mouse model were able to distinguish 5 known allergens from 5 low/non-allergens and may be useful to identify novel allergenic food proteins., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

With the aim of validating the FAMACHA(©) as a method for phenotypic selection of sheep resistant/resilient to gastrointestinal nematodes, 27 Suffolk ewes with known FAMACHA(©) score histories were experimentally infected with 25,000 larvae of Haemonchus contortus. From the day of infection (day 0) to 60 days post-infection, at intervals of 7-15 days, ewes were evaluated for packed cell volume (PCV) and fecal egg counts (FEC). A statistically significant increase (p<0.05) in FEC occurred between day 0 and day 60. PCV values showed a decrease (p<0.05) starting from day 21, compared to day 0. Based on the changes in FEC and PCV values from day 0 to day 60, 15 ewes (55.56%) were classified as susceptible (S) and 12 ewes (44.44%) as resistant/resilient (RR). A comparison of the average FEC after infection between susceptible (4487.6 eggs per gram-epg) and resistant/resilient (1317.9epg) ewes showed a significant difference (p<0.05) between the two groups. The difference in average PCV values after infection (24.8% and 30.3% for S and RR, respectively) was also significant (p<0.05). Data from 980 previous evaluations of FAMACHA(©) scores from the 27 ewes showed that 58.33% of the ewes classified as RR and 46.67% of the S group had a history of only F1 and F2 scores. In the RR group, only one animal (8.33%) had an F4 score, occurring one time out of the 61 evaluations of this ewe. In contrast, 40.0% of S group ewes had F4 and/or F5 scores. During the period of FAMACHA(©) score history that was evaluated, 69.56% of the total number of anthelmintic treatments in the flock were administered to ewes from group S. Since ewes with F4 and/or F5 scores during the FAMACHA(©) score time period were classified as susceptible during the experimental infection (with the exception of one ewe), we conclude that the FAMACHA(©) score history is a useful tool for the selection of ewes that are resistant/resilient, as well as for the identification of susceptible animals that should be culled., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Stereophotogrammetry is a radiation-free method for monitoring skull development after craniosynostosis repair. Lack of clear fixed reference points complicate longitudinal comparison of 3D photographs. Therefore we developed the 'computed cranial focal point' (CCFP)., Methods: The CCFP was calculated in segmented 3D CT-scans of 36 adult subjects using Matlab. The robustness of the CCFP calculation was evaluated in predefined hemi-ellipsoid shapes. Finally we used the CCFP in two clinical cases to correlate CT data with 3D-photographic data., Results: The CCFP calculation was found to be hardly influenced by incomplete or deformed surface data which resulted in small deviations (<2.5 mm). The average position of the CCFP of the skin relative to the sella turcica was at (0.0, 27.1, 19.4) mm, with CCFPσ (0.6, 4.6, 3.9) mm. The mean difference between the CCFP for the skull and skin was (-0.1, 1.9, -1.4) mm, with CCFPσ (0.5, 1.4, 1.0) mm. Using the CCFP in two cases to correlate the skin from a 3D-photo and the segmented skin from a CT-scan resulted in absolute mean differences of 0.7 and 2.3 mm, with a standard deviation of 1.1 mm in both cases., Conclusion: The CCFP calculation is a robust method to define a reference point relative to the sella turcica based on the skin or cranial bone surfaces. The CCFP can be used to correlate 3D photographs with CT-scan data or for longitudinal radiation-free comparison of 3D-photos., (Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

A CE-MS method has been developed to detect trace levels of potentially genotoxic alkyl halides. After derivatization of the target components with 4-dimethylaminopyridine (DMAP) or butyl 1-(pyridinyl-4yl) piperidine 4-carboxylate (BPPC), the natively positively charged derivatives are pre-concentrated by applying electrokinetic injection and separated by a highly efficient CZE method using a background electrolyte (BGE) consisting of 100mM of TRIS adjusted to pH 2.5 with phosphoric acid. Using a sheath liquid interface, subsequent MS detection allows highly specific and sensitive analysis of alkyl halides. Conditions for electrokinetic injection were optimized to allow selective and effective injection. Injection of samples with low water content at 10 kV for 150 s using a high concentration of buffer in the BGE resulted in optimum sample stacking during injection and a highly efficient CE separation. At the sample pH applied, neutral and negatively charged components are shown to be selectively discarded, resulting in injection of positively charged ions only. The sample matrix influences the efficiency of the injection, but when using an internal standard, reproducibilities better than 10% RSD are obtained. Relative recoveries of the derivatives spiked to different types of model API between 85 and 115% demonstrate that the method can be applied for quantitative analysis. Detection limits of lower than 1 mg kg(-1) for the tested alkyl halides obtained in CE-MS at least equal the sensitivity obtained in LC-MS. The CE-MS method is a valuable alternative for the LC-MS method used for analysis of alkylation compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Drug-protein conjugates have been widely used for the cell-specific targeting of drugs to cells that can bind and internalize the proteinaceous carrier. For renal drug targeting, lysozyme (LZM) can be used as an effective carrier that accumulates in proximal tubular cells. We used capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS) for the characterization of different drug-LZM conjugates. A recently developed prototype porous tip sprayer was employed for sheathless electrospray ionization (ESI) CE-MS interfacing. In order to prevent adsorption of LZM conjugates to the capillary wall, a positively charged polyethylenimine capillary coating was used in combination with a low-pH background electrolyte. Drug-LZM products had been prepared by first coupling BOC-l-methionine hydroxysuccinimide ester (BOCmet) to lysine residues of LZM followed by conjugation with the kinase inhibitors LY364947, erlotinib, or Y27632 via a platinum(II)-based linker. CE-TOF-MS of each preparation showed narrow symmetrical peaks for the various reaction products demonstrating that drug-LZM conjugates remained stable during the CE analysis and subsequent ESI. Components observed in the drug-LZM products were assigned based on their relative migration times and on molecular mass as obtained by TOF-MS. The TOF-MS data obtained for the individual components revealed that the preparations contained LZM carrying one or two drug molecules, next to unmodified and BOCmet-modified LZM. Based on relative peak areas (assuming an equimolar response for each component) a quantitative conjugate profile could be derived for every preparation leading to drug loading values of 0.4-0.6 mol drug per mole protein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: Radiofrequency ablation (RFA) has shown to improve survival in patients not eligible for surgical resection of colorectal liver metastases. However, recurrences after RFA are a major problem. Adjuvant radioimmunotherapy (RIT) after surgical resection of liver metastases has shown to improve survival. The aim of the present study was to test the hypothesis that adjuvant RIT might be an effective way to prevent recurrent liver metastases after RFA in an experimental model., Methods: Tumours in the liver were induced by intrahepatic injection of 300,000 CC531 cells in male Wag/Rij rats (n = 60). Ten days later, the intrahepatic tumours were treated with RFA. Adjuvant RIT ((177)Lu-labelled monoclonal antibody MG1 at 300 MBq/kg) was administered intravenously either at the day of RFA (day 10) or 7 days later. Control rats received no treatment. Primary endpoint was survival., Results: Administration of (177)Lu-MG1 resulted in a transient decrease in body weight, compared to no adjuvant treatment. However, no other signs of clinical discomfort were registered. Log rank test showed that the survival curves of the groups treated with RIT, either at day 10 or day 17, did not differ significantly from the survival curve of the rats that did not receive adjuvant treatment (P = 0.902)., Conclusion: This study shows that adjuvant RIT does not increase survival after RFA of colorectal liver metastases in rats., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: Hospitals in The Netherlands have recently made certain performance data public, allowing patients to choose the location of their care. The objective of this study is to assess (a) patient preferences and experiences concerning the transition between primary and secondary health care, (b) patients' needs for choice and information and how these are influenced by personal and morbidity factors., Methods: Two different types of questionnaires were used. The first questionnaire concerns the importance that patients attach to the care provided. The second questionnaire concerns the actual experiences of the patient with the care provided. For the selection of patients, we used the databases of the registration networks of the Departments of General Practice of the Universities of Groningen and Leiden. The questionnaires were returned by 513 patients (Importance 69%) and 1404 patients (Experience 65%)., Results: Many patients prefer the GP advising them regarding which hospital or specialist they should be referred to: a quarter of the patients preferred that the GP decided for them. Patients with a curable condition and patients aged between 25 and 65, highly educated and with stable personal characteristics as measured by a purposive scale, more often wished to use information from internet or newspapers to make a decision. The amount of information that was needed on illness or treatment varied greatly. Young people, older people, and those with less stable personal characteristics more often desired only practical information., Conclusions: In spite of making performance data of different health care institutions public, only a limited number of patients want to use this information on a limited number of health problems., Practice Implications: Care providers should take differences into account concerning patients' need for information on their illness., (2009 Elsevier Ireland Ltd. All rights reserved.)

A known side effect of aminoglycoside antibiotics is the development of permanent hearing loss. As South Africa is currently facing a tuberculosis (TB) epidemic, with an increasing number of multi-drug resistant tuberculosis (MDR-TB) infections, the use of aminoglycosides is on the increase. It is therefore important to determine whether the mitochondrial mutations associated with aminoglycoside-induced hearing loss occur at high frequencies in particular ethnic groups in our population. A total of 115 mainly MDR-TB patients all on aminoglycosides and 439 controls representative of the main ethnic groups in South Africa were screened for six mutations using the SNaPshot technique. Furthermore, the mitochondrial genomes of eight patients with ototoxicity were sequenced. Homoplasmic mutations were found in controls (A1555G in 0.9% of Black controls and A827G in 1.1% of Afrikaner controls) which reveal that a significant proportion of the South African population is genetically predisposed to developing aminoglycoside-induced hearing loss. The 961 delT+insC((n)) and T961G variants were found at frequencies of >1% indicating that both are probably non-pathogenic polymorphisms. Sequencing of the entire mitochondrial genome in eight patients did not reveal any mutations in the MT-RNR1 gene. However, two potentially pathogenic variants, T10114C (I19T in MT-ND3) and T15312C (I189T in MT-CYB) were found that may impact on the oxidative phosphorylation capacity and warrant further investigation for their possible role in this disorder. It is imperative that the genetic basis of this potentially preventable condition be investigated, particularly in countries where aminoglycosides are still commonly used, in order to identify individuals and/or ethnic groups who are at risk for this type of hearing loss., (Copyright 2010 Elsevier Inc. All rights reserved.)

Values of travel time savings are often used in cost-benefit analysis of transport projects and policies, and also to compute generalised travel costs. There has been considerable debate as to whether different research methods (e.g. stated versus revealed preference) will lead to different values of travel time savings, and which segmentations (e.g. by income or mode) are most important to capture the heterogeneity in these values. In addition there are many countries where no specific valuation studies have been done. In this paper new equations are estimated on the outcomes of value of travel time savings studies from various countries. In the data set, several countries appear more than once, which is taken into account by estimating random effects panel models. The meta-analysis sheds some new light on the variation of the value of travel time savings by income, country, travel purpose, mode, distance and by survey method. Furthermore, the resulting meta-models are applied to produce new values of travel time savings for business travel, commuting and for other purposes in passenger transport, for 25 European Union Member states. Similar methods could be used to statistically analyse studies carried out on other non-monetary effects, both for transport and non-transport projects, and for inclusion in cost-benefit analysis.

Objective: Development and validation of a questionnaire that measures patients' experiences of collaboration between general practitioners (GPs) and specialists., Methods: A questionnaire was developed using the method of the consumer quality index and validated in a cross-sectional study among a random sample of patients referred to medical specialists in the Netherlands. Validation included factor analysis, ascertain internal consistency, and the discriminative ability., Results: The response rate was 65% (1404 patients). Exploratory factor analysis indicated that four domains could be distinguished (i.e. GP Approach; GP Referral; Specialist; Collaboration). Cronbach's alpha coefficients ranged from 0.51 to 0.93 indicating sufficient internal consistency to make comparison of groups of respondents possible. The Pearson correlation coefficients between the domains were <0.4, except between the domains GP Approach and GP Referral. All domains clearly produced discriminating scores for groups with different characteristics., Conclusions: The Consumer Quality Index (CQ-index) Continuum of Care can be a useful instrument to assess aspects of the collaboration between GPs and specialists from patients' perspective., Practice Implications: It can be used to give feedback to both medical professionals and policy makers. Such feedback creates an opportunity for implementing specific improvements and evaluating quality improvement projects., (2009 Elsevier Ireland Ltd.)

Objective: It is well-known that vasodilatator function is affected in patients with renal failure. We hypothesized impaired venous forearm distensibility in haemodialysis patients. The purpose of this study was to investigate which provocation method generated 'maximal' venous distensibility in the forearm of haemodialysis patients compared to healthy volunteers by using duplex ultrasound., Design: The study group consisted of haemodialysis patients (n=30) and healthy volunteers (n=30). In each participant ultrasound measurements of the venous diameter were performed by using 3 different provocation methods., Methods: The applied provocation methods were: 1) hydrostatic pressure, 2) venous congestion and 3) hydrostatic pressure and warmth. Significance of differences in mean diameter changes within the groups was assessed with the paired t-test. Significance of differences in mean diameter changes between the groups was compared by using multivariate regression analysis., Results: In haemodialysis patients, the increase in mean diameter after the different methods was: 29% after methods 2 versus 1, 23% after methods 3 versus 2 and 59% after methods 3 versus 1. In healthy volunteers, the mean diameter increase was: 27% after methods 2 versus 1, 29% after methods 3 versus 2 and 64% after methods 3 versus 1. The greatest increase in the mean internal venous diameter among the haemodialysis patients and the healthy volunteers was after the provocation method which combined hydrostatic pressure with warmth (mean difference: 1mm, 95% CI: .57, 1.36; P<.001 and mean difference: 1.4mm, 95% CI: .88, 1.78; P<.001, respectively). After adjustment for the baseline variables, both groups demonstrated a non-significant mean diameter difference for each of the provocation methods., Conclusion: Hydrostatic pressure combined with warmth generates the greatest venous distensibility in the lower arm in haemodialysis patients in a sitting position and is not significantly different compared to healthy volunteers. Without the superior provocation method, venous diameters of haemodialysis patients can be assessed as false-negatives yielding that a primary radio cephalic arteriovenous fistula (RCAVF) at wrist level (the first choice) in these patients will be withheld.

The most common mutation associated with aminoglycoside-induced deafness is A1555G and it has been found in diverse populations worldwide. In the present study we investigated a large South African family known to harbour A1555G. A total of 97 family members were genotyped using the SNaPshot technique and 76 were found to be A1555G-positive (on haplogroup L0d) and are therefore at risk of developing irreversible hearing loss. The method worked equally well on both blood (from adults) and buccal swabs (from children). Variants in the tRNA(Ser(UCN)), A10S in TRMU and 35delG in GJB2 genes were shown not to act as genetic modifiers in this family. It is important to identify mutation-positive individuals and inform them of their increased risk of developing aminoglycoside-induced deafness especially in a setting like South Africa where these drugs are still commonly used because of their efficacy and cost-effectiveness as a treatment for resistant forms of tuberculosis.

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R(2)>0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N=3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.

The developments in capillary isoelectric focusing (cIEF) over the period 2003-2007 are reviewed. With the focus on technological aspects, cIEF papers published in the fields of methodology, new techniques, detection, multidimensional systems, miniaturization and applications are summarized. The methodology section covers recent research in ampholytes composition, detergents and other additives, carrier ampholyte free cIEF, coatings and other capillary modifications. In the section on new systems adjustments to the technique (e.g. dynamic IEF), different applications of cIEF (e.g. as injection system) and new devices are reported. Systems focusing on whole column imaging, fluorescence and chemiluminescence detection and coupling to mass spectrometers are discussed in the section on detection. Interfacing cIEF with MS via RPLC systems and hyphenation of cIEF with capillary electrochromatography and other capillary electrophoresis modes are also summarized. Papers focusing on miniaturization are reviewed in the section on microfluidic devices. The section on applications will show analysis of biopharmaceutical compounds and isolated proteins for metabolomic studies. For the analysis of complex biological matrices, generally multidimensional systems are needed, which are mentioned throughout this review.

On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.

A generic approach has been developed for coupling capillary electrophoresis (CE) using non-volatile background electrolytes (BGEs) with mass spectrometry (MS) using a sheath liquid interface. CE-MS has been applied for basic and bi-functional compounds using a BGE consisting of 100 mM of TRIS adjusted to pH 2.5 using phosphoric acid. A liquid sheath effect is observed which may influence the CZE separation and hence may complicate the correlation between CE-UV and CE-MS methods. The influence of the liquid sheath effect on the migration behavior of basic pharmaceuticals has been studied by simulation experiments, in which the BGE outlet vial is replaced by sheath liquid in a CE-UV experiment. As a consequence of the liquid sheath effect, phosphate based BGEs can be used without significant loss of MS sensitivity compared to volatile BGEs. The use of buffer constituents such as TRIS can lead to lower detection limits as loss of MS sensitivity can be compensated by better CE performance. TRIS based BGEs permit relatively high injection amounts of about 100 pmol while maintaining high resolution. The ESI-MS parameters were optimized for a generic method with maximum sensitivity and stable operation, in which the composition of the sheath liquid and the position of the capillary were found to be important. Furthermore, the nebulizing pressure strongly influenced the separation efficiency. The system showed stable performance for several days and a reproducibility of about 15% RSD in peak area has been obtained. Nearly all test compounds used in this study could be analyzed with an MS detection limit of 0.05% measured in scan mode using extracted ion chromatograms. As a result, CE-MS was found to be a valuable analytical tool for pharmaceutical impurity profiling.

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.

The characteristics of automated on-line solid phase extraction with liquid chromatography-mass spectrometry (SPE-LC-MS) are very amenable for flexibility and throughput in therapeutic drug monitoring (TDM). We demonstrate this concept of automated, on-line SPE-LC-MS for the analysis of clozapine and metabolites (desmethylclozapine and clozapine-N-oxide) in serum. Method development, optimisation and validation are described and a comparison with previously published methods for the determination of clozapine and metabolites in serum and plasma is made. Optimisation of chromatographic and SPE conditions for increased throughput resulted in SPE-LC-MS cycle times of only about 2.2 min, demonstrating the great potential of automated on-line SPE-LC-MS for TDM. The new method is shown to be clearly favourable, in particular in terms of ease of sample handling, throughput and detection limits. Recovery is essentially quantitative. Detection limits are at about 0.15-0.3 ng ml(-1), depending on the ionisation source used. Calibration follows a quadratic model for clozapine and its N-oxide and a linear model for the desmethyl metabolite (all cases: R > 0.99). Accuracy, evaluated at three concentration levels spanning the whole therapeutic range, shows that bias is less than 10%. Precision (intra - and inter assay) ranges from about 5% R.S.D. at the high end of the therapeutic range (700-1,000 ng ml(-1)) to about 20% R.S.D. (OECD defined limit) at the lower limit of quantitation ( approximately 50 ng ml(-1)). The lower limit of quantitation is well below the low end of the therapeutic range at 350 ng ml(-1).

In this paper, augmentation has been applied to data matrices, which originate from hyphenated methods that share the same mode of detection, but use different separation methods, HPLC-DAD and MEKC-DAD. A novel method, wavelength shift eigenstructure tracking (WET), has been proposed for the alignment between the wavelength scale of both detectors. WET proves to be suitable for the detection as well as correction of wavelength shift between both detectors. After correction of the wavelength scale, data obtained on both systems have been augmented and submitted to iterative target transformation factor analysis. Augmented curve resolution provides significantly better estimates of the chromatographic and electrophoretic profiles and spectra than the use of non-augmented curve resolution on HPLC and MEKC data separately. It is particularly useful when the pure fraction of a chromatographic peak is less than 0.10. Finally, the relative weight of MEKC versus HPLC in augmentation may be increased using intensity and noise normalisation. However, since noise normalisation and its accompanying decrease in signal-to-noise ratio leads to a loss of information, and, since intensity normalisation may cause a failure of the augmented curve resolution algorithm, benefits and drawbacks of normalisation should be weighed on a case-by-case basis.

Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.

The aim of this article was to study mortality following a first-ever cerebral infarct, accounting for ischemic stroke subtypes (lacunar, cardioembolic, atherothrombotic) and relevant prognostic variables. This study was done from s a hospital-based prospective registry of all patients with a first cerebral infarct, with a high case ascertainment of first and recurrent stroke by CT. We used a cross-sectional follow-up, using standardized methods. Analyses were performed using crude comparison of mortality data and death causes between stroke subtypes. We analyzed 30-day case fatality and 1-year mortality in 30-day survivors by means of logistic regression analysis, and mortality in 1-year survivors by means of Cox proportional hazard modeling. We also constructed Kaplan-Meier survival curves, and used log-rank testing for differences between stroke subtypes. Thirty-day case fatality was 10%, 1-year mortality 15%, and after 1-year mortality 16%. Mean follow-up was 691, SD 521 days. At the end of follow-up 36% of all patients had died. Mortality was at all three time points lowest in lacunar stroke (2, 12, and 14%, respectively), intermediate in atherothrombotic stroke (10, 16, and 15%, respectively), and highest in cardioembolic stroke (23, 22, and 21%, respectively). Death related to recurrent stroke was similar in all three stroke subtypes (13-16%). Although 30-day case fatality rate was low in lacunar stroke, a quarter of lacunar stroke patients had died at the end of follow-up. Diabetes mellitus, age, stroke subtype, and initial stroke severity were independent predictors of 30-day case fatality, but only diabetes and age were consistent independent predictors for later mortality. Recurrent stroke and heart failure were important death causes. Prognosis for (future) death following a first cerebral infarct differs between stroke subtypes; lacunar stroke patients have the lowest mortality. However, lacunar stroke cannot be regarded as a mild stroke type, as after 2 years more than a quarter of such stroke patients had died. Cardioembolic stroke patients have the grimmest prognosis: more than half of them had died within 1.5 years. Better prognosis for long-term survival following stroke may be achieved by therapies which lower the risk of stroke recurrence, provide better treatment of heart failure, or both.

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.

The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 microM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping., (Copyright 2002 Elsevier Science BV.)

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

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.

The reversed-phase liquid chromatographic analysis of basic pharmaceuticals can be problematic. Both the properties of the eluent and the stationary phase can influence the chromatographic performance. Therefore selection of suitable experimental conditions for the analysis of basic compounds can be difficult. This paper shows that the organic modifier and the nature of the buffer influence the eluent properties. Moreover, the nature and amount of modifier also influence the basicity of the analytes. Investigations showed that the nature of the buffer can have a significant influence on retention and peak shape of basic compounds. Test procedures using basic analytes as test probes provided relevant information with respect to selecting columns to analyse basic pharmaceutical compounds. Test procedures using compounds like aniline, phenol and benzene were found to be less suitable.

Recent reports suggest that the radiation-induced, p53-dependent, apoptotic response is aberrant in ataxia telangiectasia (AT) cells. We investigated the possibility that an aberrant apoptotic response to ionizing radiation may also be the characteristic of AT heterozygotes and may facilitate in discriminating AT heterozygotes from the general population. Log phase, Epstein Barr virus (EBV) transformed lymphoblastoid cell lines and primary lymphocytes from three AT families were irradiated and the apoptotic response at 30h post radiation was measured by flow cytometry using TUNEL and hypodiploid methods. Our results show that the apoptotic response of AT homozygote (ATM-/-), AT heterozygote (ATM+/-) and normal cells (ATM+/+) to ionizing radiation, measured by the hypodiploid and TUNEL methods using flow cytometry, is dose and time dependent. Furthermore, this response is paradoxical in that ATM (-/-) lymphoblastoid cells were characterized by a reduced post radiation apoptotic response compared to their normal counterparts. Heterozygote (ATM+/-) lymphoblastoid cells displayed an intermediate response to ionizing radiation. In contrast, primary, non-transformed AT cells exhibited the same apoptotic response as their normal counterparts. Our results thus indicate that pre-radiation, EBV-transformed, lymphoblastoid cell lines from individual families may be useful in discriminating ATM status, but patient-derived, primary AT homozygous, heterozygous and normal primary cultured lymphocytes cannot be discriminated by this assay.

Placental alkaline phosphatase (PLAP) that had been isolated from human placenta was further purified using subsequent ion-exchange chromatography (IEC), affinity chromatography (AC) and centrifugal membrane concentration (CMC). During the process, the PLAP samples from the different stages of purification were characterized regarding purity and activity. This was accomplished by combining Lowry analysis, enzymatic activity assay, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS). The sample obtained after IEC had a rather low specific activity (6.8 U/mg) and appeared to contain several major contaminants, among which was human serum albumin (HSA). AC followed by CMC yielded PLAP with a specific activity of 128 U/mg. The purity and identity of the protein was indicated by MALDI-TOF-MS yielding a spectrum with one major peak at m/z 58,101. Interestingly, CZE of the pure PLAP revealed a cluster of peaks, which probably reflects the presence of various glycoforms and/or oligomers. The same analytical approach was used to characterize commercially available PLAP. This sample showed a moderate specific activity (15 U/mg) and appeared to be highly impure containing various other proteins.

Chronic hypertension during aging is a serious threat to the cerebral vasculature. The larger brain arteries can react to hypertension with an abnormal wall thickening, a loss of elasticity and a narrowed lumen. However, little is known about the hypertension-induced alterations of cerebral capillaries. The present study describes ultrastructural alterations of the cerebrocortical capillary wall, such as thickening and collagen accumulation in the basement membrane of aging spontaneously hypertensive stroke-prone rats. The ratio of cortical capillaries with such vascular pathology occurred significantly more frequently in hypertensive animals. Nimodipine and nifedipine are potential drugs to decrease blood pressure in hypertension but their beneficial effects in experimental studies reach beyond the control of blood pressure. Nimodipine and nifedipine can alleviate ischemia-related symptoms and improve cognition. These drugs differ in that nifedipine, but not nimodipine reduces blood pressure at the here-used concentration while both drugs can penetrate the blood-brain barrier. Here we show that chronic treatment of aging hypertensive stroke-prone rats with nimodipine or nifedipine could preserve microvascular integrity in the cerebral cortex.

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.

Solid-phase microextraction (SPME) has been introduced for the extraction of organic compounds from environmental samples. This relatively new extraction technique has now also gained a lot of interest in a broad field of analysis including food, biological and pharmaceutical samples. SPME has a number of advantages such as simplicity, low cost, compatibility with analytical systems, automation and the solvent-free extraction. The last few years, SPME has been combined with liquid chromatography and capillary electrophoresis, besides the generally used coupling to gas chromatography, and has been applied to various biological samples such as, e.g., urine, plasma and hair. The objective of the present paper is a survey of the application of SPME for the analysis of biological samples. Papers about the analysis of biologically active compounds are categorised and reviewed. The impact of SPME on various analytical fields (toxicological, forensic, clinical, biochemical, pharmaceutical, and natural products) is illustrated. The main features of SPME and its modes are briefly described and important aspects about its application for the determination of pharmaceuticals, drugs of abuse and compounds of clinical and toxicological interest are discussed. SPME is compared with other sample pretreatment techniques. The potential of SPME and its main advantages are demonstrated. Special attention is paid to new trends in applications of SPME in bioanalysis.

Theoretical aspects of multiple solid-phase microextraction are described and the principle is illustrated with the extraction of lidocaine from aqueous solutions. With multiple extraction under non-equilibrium conditions considerably less time is required in order to obtain an extraction yield that is equal to that of one extraction at equilibrium. On the other side, the extraction yield can be increased if multiple extraction is performed with the same total time as is needed for one extraction at equilibrium time. The effect of multiple extraction is strongly dependent on the value of the partition constant and for practical use the length of the desorption time is important. A good agreement between theoretical and experimental data has been obtained. Chromatograms are presented showing the potential of multiple solid-phase microextraction.

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.

Direct-immersion solid-phase microextraction (SPME) has been used to extract the local anesthetic lidocaine from human plasma. A simplified model shows the relationship between the total amount of drug in plasma and the amount of drug extracted. The model takes into account that the drug participates between the fiber, sample and proteins. Therefore the model can also be used to obtain a good approximation of the drug-protein binding. Extraction yields of lidocaine in plasma are <1%, and the protein binding of lidocaine was found to be about 74% at pH 9.5. A SPME method has been developed for the determination of the total amount of lidocaine in plasma. The protein binding was reduced by acidification and, subsequently, the sample was deproteinized with trichloroacetic acid. With a 100-microm polydimethylsiloxane-coated fiber and addition of sodium chloride to the sample an extraction yield of about 12% at equilibrium (45 min) has been obtained. The relative standard deviation of this method is <10%. A linear range was found from 25 to 2000 ng ml(-1) lidocaine in plasma (r=0.998) with a detection limit of 5 ng ml(-1) in plasma. An extraction yield of about 80% could be obtained after an overnight extraction by use of a 65-microm polydimethylsiloxane-divinylbenzene-coated fiber. If an extraction time of 10 min is used with this fiber, the same yield is obtained as with the single-phase fiber in 45 min. However, the drawback of this mixed-phase fiber is its much shorter lifetime.

Theoretical and empirical models can be used to model the migration or separation characteristics in micellar electrokinetic chromatography in order to optimise the resolution. In this paper only empirical models were used, because it is easier and more straightforward to obtain these models. Several empirical approaches for the optimisation of the resolution were compared in order to determine which response should be modelled preferably. The use of models of the effective mobility in combination with average plate numbers proved to be the most suitable approach to optimisation of the resolution, because the relative prediction errors of the models of the effective mobility were a factor of 2-4 smaller than the relative prediction errors of the models of the apparent mobility. Moreover for the least separated peak pair the resolutions based on the models of the apparent and effective mobility showed relative prediction errors that were approximately a factor of 2 smaller than the relative prediction errors of the resolutions based on the models of the resolution and separation factor. The predictions of the separation factor based on the different models generally showed lower prediction errors than the predictions of the corresponding resolutions. Although the relative prediction errors were large, particularly for closely migrating compounds, the empirical approach will probably lead to the optimum separation buffer composition.

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

Solid-phase extraction-pipette tips were used for micro solid-phase extraction of lidocaine and diazepam. Off-line desorption was done after in-vial collection for reference purposes, whereas with on-line desorption the eluate was directly introduced in the gas chromatograph. With both methods the total eluate (100 microl) was introduced into the GC system, which was equipped with a programmed-temperature vaporiser (PTV) for large volume injection. For on-line desorption a laboratory-made coupling device was developed to connect the pipette tips with the injector of the PTV. The coupling device was applied successfully since no leakage occurred at the connection of the coupling device and the pipette tip. No significant differences in recovery of lidocaine and diazepam and in presence of impurities were observed between chromatograms obtained with either off-line or on-line desorption. Preliminary experiments with standard solutions showed recoveries of about 75% for a concentration level of 1 microg/ml. The system seems particularly suitable for high-throughput analysis.

The use of a programmed temperature vaporiser (PTV) with a packed liner was evaluated for the injection of large volumes (up to 100 microl) of plasma extracts in a gas chromatograph. Solvent purity, which is essential when large volumes are injected into the GC system, was determined. Special attention was paid to the purity of the solvents used for the solid-phase extraction (SPE) procedure. For this SPE method, ethyl acetate was used as the extraction and reconstitution solvent, and thus the purity of the ethyl acetate was critical, especially when a non-selective GC detector was applied. The liquid capacity and inertness of different packed liners were investigated. The liner packed with ATAS "A" (modified Chromosorb-based material with special treatment) was found to be the most suitable for the analysis of the tested drugs. Good linearity in response for variations in volume and concentration was observed. A comparison was made between the applicability of flame ionisation detection (FID) and mass-selective detection (MSD). When 50-microl volumes of plasma extracts were injected with the PTV, the detection limits for secobarbital, lidocaine, phenobarbital and diazepam were about 50-times lower than when 1-microl volumes were injected. The detection limits of the tested compounds in plasma for injection of 50-100 microl plasma extract are 5-10 ng/ml for GC-FID whereas plasma concentrations of 250 pg/ml can be detected using the selected ion monitoring (SIM) mode of a MSD. For non-selective GC-FID, the background from a 50-microl injection was substantially larger than with 1-microl injection as a result of co-injected plasma matrix components and solvent impurities. These background effects were less with GC-MSD in the total ion current mode and virtually absent with GC-MSD in the SIM mode.