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In this instalment of 'Sample Preparation Perspectives,' Norwegian authors from the University of Oslo describe the practical aspects of hollow fibre liquid-phase microextraction in the three-phase mode [(HF.sup.3]LPME). The guest [...]

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Background: Although the mortality of coronary heart disease (CHD) has declined in Western countries during the last decades, studies have suggested that the prevention and treatment of CHD may not have been as effective in women as in men. We examined gender- and age-specific trends in incidence, case fatality and the severity of first myocardial infarction (MI) in a large Norwegian population-based study.Design: Prospective population-based cohort study.Methods: A total of 31,323 participants enrolled between 1974 and 2001 were followed throughout 2004 for a total of 400,572 person-years. Suspected coronary events were adjudicated by a review of hospital records and death certificates. A total of 1669 events fulfilled standardized criteria of first-ever fatal or non-fatal MI.Results: In the age group 35–79 years, the age-adjusted incidence of MI declined significantly in men, whereas an increase was observed in women. For men and women ≥80 years the incidence rates remained unchanged. The severity of MI and the 28-day and 1-year case fatality rates declined significantly and similarly in men and women.Conclusion: Trends in MI incidence differed by sex and age; in the age group 35–79 years a marked decrease was observed among men but an increase was observed among women, while no change was observed among older patients. MI severity and case fatality were clearly reduced for both sexes. These data suggest that the burden of CHD is shifting from middle-aged men toward middle-aged women and elderly patients.

In this instalment of "Sample Preparation Perspectives," Norwegian authors from the University of Oslo describe the practical aspects of hollow fibre liquid-phase microextraction in the three-phase mode (HF³LPME). The guest authors highlight important practical issues related to the supported liquid membrane, the hollow fibre and the extraction itself. They also discuss practical work with electromembrane extraction (EME), which is related to HF³LPME but uses an electrical potential as the driving force for the extraction. [ABSTRACT FROM AUTHOR]

Electro-assisted extraction of ionic drugs from biological fluids through a supported liquid membrane (SLM) and into an aqueous acceptor solution was recently introduced as a new sample preparation technique termed electromembrane extraction (EME). The applied electrical potential across the SLM has typically been in the range of 1 - 300 V. Successful extractions have been demonstrated even with common batteries (9 V) instead of a power supply. The chemical composition of the SLM has been crucial for the selectivity and for the recoveries of the extraction. Compared to other liquid-phase microextraction techniques (LPME), extraction times have been reduced by a factor of 6 - 17, and successful extractions have been obtained at extraction times of 1 - 5 min, and even down to a few seconds with online microfluidic EME devices. The technique has provided very efficient sample clean-up and has been found well suited for the extraction of sample sizes in the low µL range. Extractions have been performed with both rod-shaped hydrophobic porous fibers and with flat hydrophobic porous sheets as SLM support. The technique has been successfully downscaled into the micro-chip format. The nature of the SLM has been tuned for extraction of drugs with different polarity allowing extractions to be tailored for specific applications depending on the analyte of interest. The technique has been found to be compatible with a wide range of biological fluids and extraction of drugs directly from untreated human plasma and whole blood has been demonstrated. EME selectively extracts the compounds from the complex biological sample matrix as well as allowing concentration of the drugs. With home-built equipment fully acceptable validation results have been obtained.

Thirty‐five different basic drugs were extracted by electro membrane extraction (EME), from acidified samples containing HCl as the BGE, through an organic solvent immobilized in the pores in the wall of a porous hollow fiber (supported liquid membrane, SLM), and into an acidified acceptor solution (HCl) in the lumen of the hollow fiber by the application of an electrical potential difference of 50 V. With 2‐nitrophenyl pentyl ether (NPPE) as the SLM, and with 10 mM HCl as BGE in the sample and acceptor solution, singly charged basic drugs with logP>2 were extracted with recoveries in the range 30–81% within 5 min. For doubly charged basic drugs, extraction was effectively enhanced by decreasing the concentration of HCl in the sample from 10 to 0.1 mM, reducing the ionization of the analytes. For medium polar analytes (1 < logP < 2), an ion balance of 0.01 was combined with addition of tris‐(2‐ethylhexyl) phosphate (TEHP) to the SLM, and this provided recoveries in the range 36–70%. The ion balance was defined as the concentration ratio of BGE between the sample and the acceptor solution. For the most polar drugs (log P<1), EME was accomplished with an ion balance of 0.01 and with di‐(2‐ethylhexyl) phosphate (DEHP) added to the SLM, but in spite of this, recoveries were in the range of only 4–17%.

In this work, three‐phase liquid‐phase microextraction (LPME) based on a supported liquid membrane (SLM) sustained in the wall of a hollow fiber was investigated with special focus on optimization of the experimental procedures in terms of recovery and repeatability. Recovery data for doxepin, amitriptyline, clomipramine, and mianserin were in the range of 67.8–79.8%. Within‐day repeatability data for the four basic drugs were in the range of 4.1–7.7%. No single factor was found to be responsible for these variations, and the variability was caused by several factors related to the LPME extractions as well as to the final HPLC determination. Although the volume of the SLM varied within 0.4–3.1% RSD depending on the preparation procedure, and the volume of the acceptor solution varied within 4.8% RSD, both recoveries and repeatability were found to be relative insensitive to these variations. Thus, the handling of microliters of liquid in LPME was not a very critical factor, and the preparation of the SLM was accomplished in several different ways with comparable performance. Reuse of hollow fibers was found to suffer from matrix effects due to built‐up of analytes in the SLM, whereas washing of the hollow fibers in acetone was beneficial in terms of recovery, especially for the extraction of the most hydrophobic substances. Several of the organic solvents used in the literature as SLM suffered from poor long‐term stability, but silicone oil AR 20 (polyphenyl‐methylsiloxane), 2‐nitrophenyl octyl ether (NPOE), and dodecyl acetate (DDA) all extracted with unaltered performance even after 60 days of storage at room temperature.

The extractability of 58 different basic drugs by 3‐phase liquid‐phase microextraction (LPME) was studied. Extraction recoveries were correlated to solubility data and log Ddata calculated with a commercial computer program. The basic drugs were extracted from 1.5 mL water samples (pH 13) through approximately 15 μL of dodecyl acetate immobilized within the pores of a porous polypropylene hollow fibre (organic phase), and into 15 μL of 10 mM HCl (acceptor solution) present inside the lumen of the hollow fibre. Compounds with a calculated solubility below 1 mg/mL at pH 2 were poorly recovered and remained principally in the organic phase. For these drugs, 2‐phase LPME may be used as an alternative technique, where the aqueous acceptor phase is replaced by an organic solvent. In the solubility range 1–5 mg/mL, most drugs were effectively extracted (recovery > 30%), whereas drugs belonging to the solubility range 5–150 mg/mL were all extracted with recoveries above 30% by 3‐phase LPME. The hydrophilic nature of most drugs with solubilities above 150 mg/mL prevented them from entering the organic phase, and only those with log D>1.8 were effectively recovered by 3‐phase LPME. For drugs with log D< 1.8 (and solubility > 150 mg/mL), carrier‐mediated LPME was found to be the preferred technique, where an ion‐pair reagent (octanoic acid) was added to the sample. In the case of carrier‐mediated LPME, the volume of sample was decreased to 100 μL to facilitate rapid extractions. Based on the present work, the extractability of new compounds may easily be predicted to speed up method development. Extractions were also accomplished from plasma samples, where interactions between proteins and the drugs may reduce the extraction recovery. However, dilution of the plasma samples with water and adjustment of pH into the alkaline region effectively suppressed drug‐protein interactions for most of the drugs studied.

Hollow fibre based liquid‐phase microextraction (LPME) using fatty oils and essential oils as the organic phase was evaluated to develop sample preparation technology eliminating the use of hazardous organic solvents. Basic drugs were extracted from different aqueous samples (0.2 to 1 mL) through approximately 15 μL of either almond oil, arachis oil, olive oil, soy‐bean oil, anise oil, fennel oil, lavender oil, or peppermint oil (organic phase) immobilised within the pores of a polypropylene hollow fibre and into 20 μL of 10 mM HCOOH (acceptor phase) present inside the lumen of the hollow fibre. The extraction performance of the essential oils was comparable with the solvents normally used in LPME (dihexyl ether, n‐octanol, and dodecyl acetate) in terms of extraction recovery and extraction speed. Whereas all essential oils tested were compatible with human urine, only anise oil was successful for plasma. The fatty oils provided lower recoveries than the essential oils due to higher viscosity, but all the fatty oils were compatible both with urine and plasma samples. In spite of the multi‐component nature of the oils tested, they were not found to seriously contaminate the acceptor phases during extraction. In conclusion, fatty oils and essential oils may serve as alternative organic phase in LPME, eliminating the use of hazardous organic solvents.

Liquid-phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean-up and high preconcentration. The present paper describes the combination of LPME with LC-MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC-MS system. The system was qualitatively investigated for matrix effects utilising a post-column infusion system. Whole blood from 5 different persons was cleaned-up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post-column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (&lt; 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid–liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2–20 ng ml⁻¹ for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses. Copyright © 2003 John Wiley & Sons, Ltd.

Recently, we introduced an inexpensive and disposable hollow fiber-based device for liquid-phase microextraction (LPME) where ionic analytes typically were extracted and preconcentrated from 1–4 mL aqueous samples (such as plasma and urine) through an organic solvent immobilized in the pores of a polypropylene hollow fiber and into a 10–25 μL volume of acceptor phase present inside the lumen of the hollow fiber. Subsequently, the acceptor phase was directly subjected to the final analysis by a chromatographic or electrophoretic method. In the present work, attention was focused on LPME of the basic drugs amphetamine, pethidine, promethazine, methadone and haloperidol characterized by substantial differences in the degree of protein binding. Drug–protein interactions in plasma resulted in reduced recoveries and substantially increased extraction times compared with extraction of the drugs from a pure water matrix. However, by addition of 5–50% methanol to the plasma samples, recoveries were comparable with LPME from water samples and ranged between 75 and 100%. The addition of methanol was found not to speed up the LPME process and extractions from plasma were performed in 45 min to reach equilibrium. Because approximately 55–70% of the final analyte concentrations were achieved within the initial 10 min of the LPME process, validation was accomplished after 10 and 45 min of LPME. In general, the results with 10 and 45 min were almost comparable, with precision data in the range 1.2–11.1% (RSD) and with linearity in the concentration range 20–1000 ng mL−1 (r = 0.999). In conclusion, excellent LPME results may be achieved in a short time under non-equilibrium conditions with a minor loss of sensitivity. In cases of drug–protein interactions, methanol may be added to ensure a high extraction recovery.

Recently, we introduced an inexpensive and disposable hollow fibre based device for liquid‐phase microextraction (LPME) where ionic analytes typically are extracted from 1–4 mL aqueous samples (such as plasma and urine) through an organic solvent immobilized in the pores of a polypropylene hollow fibre and into a 10–25 μL volume of acceptor phase present inside the hollow fibre. Because of the substantial volume ratio of the sample relative to the acceptor phase, ionic analytes may be preconcentrated considerably during LPME (typical preconcentration factors of 50 to 150). In addition, during LPME from biological samples such as plasma and urine, the majority of matrix components are not extracted into the acceptor phase resulting in excellent sample clean‐up. For the extraction of basic drugs, dilute solutions of HCl or acidic phosphate buffers have been utilized as acceptor phase without further optimization. In the present work therefore, systematic studies have been performed to explore new acidic acceptor phase possibilities in combination with dihexyl ether as a standard organic solvent in the pores of the hollow fibre. In general, 10 mM solutions of HCl, H2SO4, and phosphate pH 3.3 were found to provide the highest extraction recoveries. pH was found to play a major role in adjusting the selectivity of LPME, whereas minor selectivity alterations were observed on changing the type of acid in the acceptor phase. For weakly basic drugs, an increased level of HCl in the acceptor phase may provide higher recovery, and for certain drugs, addition of ethanol to the acceptor phase may improve the extractability.

Liquid‐Phase microextraction based on polypropylene hollow fibres was combined with flow‐injection tandem mass spectrometry for rapid screening of drugs in biological matrices. Amphetamine and analogues were utilised as model compounds. These drugs were extracted from 0.5 mL samples of whole blood or urine. The samples were made alkaline with 0.5 mL of 1 M NaOH and the urine samples were diluted with 3 mL water to reduce the salt concentration. The uncharged analytes were then extracted through a hollow fibre impregnated with dihexyl ether into 25 μL of 0.01 M HCl inside the hollow fibre. Parallel extraction of 20–30 samples was performed for 15 min. After extraction 20 μL of the extract was injected directly into the flow‐injection tandem mass spectrometry system. Atmospheric pressure ionisation operated in positive mode was used as ion spray. All analytes were detected simultaneously after 0.1 min, utilising a combination of selected ion monitoring mass spectrometry and selected reaction monitoring tandem mass spectrometry. Limits of detection (S/N= 5) varied between the compounds and were estimated to be 2–100 ng/mL in urine and 0.4–14 ng/mL in whole blood. Comparison of injection of pure acceptor solution with urine and whole blood extracts demonstrated the efficient sample clean‐up by LPME. Ion suppression due to matrix effects was not seen, rendering LPME‐FIA‐APCI‐MS‐MS a promising alternative for rapid screening of drugs in biological matrices.

BackgroundIn epidemiological studies, the electrocardiogram has often been interpreted by means of a categorical classification. Computerized recording offers the possibility of analysing electrocardiographic measurements as continuous variables.ObjectiveTo test the hypothesis that duration of QRS complex and T-wave inversion would be independent predictors of myocardial infarction.MethodsIn a population-based study, we prospectively investigated the risk of developing myocardial infarction according to duration of QRS complex and peak-to-peak T-wave amplitude measured from lead I of the 12-lead electrocardiogram by computerized electrocardiography. In total 6628 men aged 25–61 years who had not previously suffered a myocardial infarction were followed up for 3.9 years.ResultsEighty-two first myocardial infarctions (55 non-fatal and 24 fatal myocardial infarctions and three sudden deaths) were identified. The risk of myocardial infarction increased with duration of QRS complex and with decreasing T-wave amplitude. A proportional hazards model with adjustment for possible confounders yielded a relative risk of myocardial infarction of 3.74 (P for linear trend 0.015) for duration of QRS complex ≥ 120 ms compared with duration of QRS complex < 80 ms. The multivariate relative risk for T-wave amplitude ≥ 0.35 mV compared with T-wave amplitude < 0.20 mV was 0.55 (P for linear trend 0.036). When both duration of QRS complex and T-wave amplitude were included in the multivariate model, T-wave amplitude retained its predictive power, whereas duration of QRS complex became marginally no longer significant (P = 0.067).ConclusionsPeak-to-peak T-wave amplitude from lead I is an independent predictor of myocardial infarction in men who have not previously suffered a myocardial infarction. Greater duration of QRS complex clearly indicates a higher risk of myocardial infarction. However, when T-wave amplitude is included as a covariate, the predictive power of duration of QRS complex does not remain significant Single-lead electrocardiography is a feasible method for improving the assessment of the relative risk of myocardial infarction.

Background:It is widely recognized that in some people it is difficult or impossible to acquire adequate measurements of cardiac performance and anatomy by any echocardiographic technique. We used our population-based screening to determine the characteristics of such unmeasurable subjects. Method:In a sample of 3287 subjects aged 25 to 85 years, we used standard 2-dimensional guided M-mode echocardiography and pulsed and color Doppler to assess left ventricular (LV) structure and function. Results:Of 3287 subjects only 0.4% could not be measured by any technique. In 2794 subjects M-mode registrations of good quality were obtained, which allowed calculation of LV mass and LV ejection fraction. Those in whom measurements could not be obtained had a significantly higher age, body mass index, blood pressure, waist/hip ratio, and were more likely to smoke, be a man, be taking antihypertensive medication, have a history of ischemic heart disease, and have a low level of physical activity. Conclusion:Because subjects with high cardiovascular risk factor levels are less likely to be measurable with echocardiography, a need exists for other noninvasive diagnostic methods in these persons. (J Am Soc Echocardiogr 1999;12:314-8.)

On the basis of a questionnaire in a population study in the county of Nordland, Norway, the prevalence of palpitations and its associations to some lifestyle factors, depression and self-rated health were analysed. All the 10,497 residents aged 40 to 42 years were invited to participate, 82% attended, 87% of the attenders returned a questionnaire by mail, and 6436 subjects were included in this report. The prevalence of palpitations was 15% in men and 25% in women. Palpitations were significantly associated with coffee consumption, smoking, alcohol intoxication, physical inactivity, depression and poor self-rated health in the univariate analyses. In a logistic regression analysis, the relations between palpitations and lifestyle were weakened. Significant predictors for palpitations were depression and poor self-rated health in both sexes, in addition to heavy coffee drinking and physical inactivity in men and alcohol intoxication in women. In conclusion, palpitations were more firmly linked to depression and self-evaluated health than to lifestyle.

An automated method for residue analysis of oxolinic acid and flumequine in liver of Atlantic salmon is described. Oxolinic acid and flumequine are extracted from liver with 0.4 M phosphate buffer pH 10 and the extracts are automatically analysed by on-line dialysis and column-switching in an HPLC system. The limit of detection was 4 μg/kg for oxolinic acid and 7 μg/kg for flumequine with fluorescence detection. The on-line combination of dialysis and column-switching HPLC was shown to be a reliable technique for residue control of these drugs in fish liver.

Different organic borates, phosphates, sulphates, and carboxylic acids are evaluated as extraction carriers in three-phase liquid phase microextraction (LPME). Hydrophilic basic drugs form ion pairs with the carriers and are extracted as ion-pair complexes into an organic liquid membrane of n-octanol or peppermint oil immobilized in the pores of a polypropylene hollow fiber. From this point, the basic drugs are released into a 20-µL solution of 50mM HCl placed inside the lumen of the hollow fiber (acceptor solution). Simultaneously, the carrier is neutralized by protons from the acceptor solution (protonated to maintain the charge balance). Both water-soluble and water-insoluble carriers are tested. One promising candidate among the water-soluble carriers is 1-heptanesulfonic acid. This is added to the sample solution to a final concentration of 25mM and served to ion-pair the analytes within the sample solution. Among the less water-soluble candidates, a mixture of di(2-ethylhexyl) phosphate (DEHP) and tris(2-ethylhexyl) phosphate (TEHP) serve as efficient carriers. Ten percent (w/w) of each of DEHP and TEHP are added to the organic liquid membrane, and these carriers principally worked through ion-pairing with the analytes at the interface between the sample solution and the organic liquid membrane. Several carriers are found to be compatible with human plasma samples, and bromthymol blue is particularly efficient in combination with these protein-containing matrices. Following optimization of the conditions for bromthymol blue, including saturation of the plasma samples with sodium sulphate, extraction recoveries between 45% and 75% are obtained for eight model drugs after 60 min of extraction. With bromthymol blue as the carrier, highly acceptable validation data are obtained for phenylpropanolamine and practolol extracted from human plasma.

A versatile injection system for the direct introduction of samples into the gas chromatograph (GC) is described. Several years of experience in biological and pharmaceutical chemistry have led to the construction and final modification of this device. No modification of the injection port of the GC is necessary.