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Purpose: Melatonin aids in the synchronization of the circadian rhythm to the external environment. Few studies have tried to elucidate the relationship between melatonin and obstructive sleep apnea syndrome (OSAS). These often include few patients, do not differentiate between OSAS severity and/or do not analyse a 24-h melatonin profile. This study set out to investigate disease severity dependent differences in 24-h salivary melatonin secretion of OSAS patients compared to a reference population in a retrospective design., Methods: 24-h salivary melatonin profiles of 169 OSAS patients were analysed (55 light, 66 moderate, 48 severe) as well as 91 reference patients. Several aspects of the melatonin curve were analysed and stratified according to OSAS severity. Parameters included: dim light melatonin onset (DLMO), time of returning below DLMO (DLMOoff), peak melatonin concentration and time, and total melatonin exposure., Results: Significant effects were corrected for confounding by age and sex using linear regression. Our analysis shows that, compared to reference and in a disease dependent manner, OSAS patients have a significantly lower 24-h melatonin curve, lower melatonin peak concentration, lower total melatonin exposure and a smaller proportion of patients reach DLMO. The differences in peak melatonin production and total melatonin exposure were resistant to confounding by age and/or sex., Conclusion: This study describes clear OSAS severity dependent abnormalities in melatonin production in OSAS patients, independent of sex and/or age. Future research should indicate whether oral melatonin supplementation has beneficial effects in OSAS patients with attenuated endogenous melatonin production., (© 2024. The Author(s).)

Mild traumatic brain injury (mTBI) is a common condition seen in emergency departments worldwide. Blood-based biomarkers glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) are recently U.S. Food and Drug Administration-approved for the prediction of intracranial lesions on head computed tomography (CT) scans in mTBI. We evaluated the diagnostic performance of GFAP and UCH-L1 in a Dutch cohort using the i-STAT TBI assay. In a multi-center observational study, we enrolled 253 mTBI patients. Head CT scans were scored using the Marshall classification system. Logistic regression models were used to assess the contribution of biomarkers and clinical parameters to diagnostic performance. Detection of UCH-L1 and GFAP resulted in a sensitivity of 97% and specificity of 19% for CT positivity in mTBI patients, along with a negative predictive value of 95% (88-100%) and a positive predictive value of 27% (21-33%). Combining biomarker testing with loss of consciousness and time to sample increased specificity to 46%. Combined testing of UCH-L1 and GFAP testing resulted in possibly more unnecessary CT scans compared with GFAP testing alone, with only limited increase in sensitivity. This study confirmed high sensitivity of GFAP and UCH-L1 for CT abnormalities in mTBI patients using the i-STAT TBI test. The results support the potential use of GFAP and UCH-L1 as tools for determining the indication for CT scanning in mTBI patients, possibly offering a cost- and time-effective approach to management of patients with mTBI. Prospective studies in larger cohorts are warranted to validate our findings.

Introduction: A considerable amount of neonatal morbidity and mortality worldwide is caused by preterm birth. To date, the underlying etiology of preterm birth has not been fully clarified. Previous studies demonstrate that inflammation is one of the pathological factors that might cause preterm birth, and that there is a difference between primiparous and multiparous women in immune response to pregnancy. The objective of this prospective cohort study was to investigate the role of two inflammatory markers, ferritin and C-reactive protein (CRP) and preterm birth, in first trimester women, stratified for parity. In addition, a possible association between high ferritin and CRP, and a possible association between high ferritin and CRP and preterm birth were assessed., Material and Methods: A total of 2044 healthy, low-risk pregnant women from primary obstetric care in the Netherlands participated in this study. Their ferritin and CRP levels were evaluated at 12 weeks' gestation. Levels above the parity specific 95th percentile were defined as high. The main outcome of this study was to assess the presence of a possible association between parity specific high ferritin and CRP, and preterm birth. The secondary outcomes were the ferritin and CRP levels of women, stratified for parity, and the possible association between high ferritin and CRP levels. Logistic regression analysis was performed with preterm birth as a dependent variable and parity specific high ferritin and CRP as an independent variable, adjusting for age and history of preterm birth., Results: Ferritin levels decreased with increasing parity. Ferritin and CRP levels at 12 weeks' gestation were significantly higher in women with preterm birth. In primiparous women, high ferritin levels (OR: 2.5, CI: 1.14-5.38) and high CRP levels (OR: 5.0, CI: 2.61-9.94) were independently associated with preterm birth. In multiparous women, high ferritin levels (OR: 6.0, CI: 2.28-16.67) were independently associated with preterm birth while high CRP levels were not., Conclusions: First trimester parity specific ferritin and CRP levels could play a part in predictive models for preterm birth, and further research for their additive role in preterm birth is needed., (© 2024 The Authors. Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

The Contrast Media Safety Committee of the European Society of Urogenital Radiology has, together with the Preanalytical Phase Working Group of the EFLM Science Committee, reviewed the literature and updated its recommendations to increase awareness and provide insight into these interferences. CLINICAL RELEVANCE STATEMENT: Contrast Media may interfere with clinical laboratory tests. Awareness of potential interference may prevent unwanted misdiagnosis. KEY POINTS: • Contrast Media may interfere with clinical laboratory tests; therefore awareness of potential interference may prevent unwanted misdiagnosis. • Clinical Laboratory tests should be performed prior to radiological imaging with contrast media or alternatively, blood or urine collection should be delayed, depending on kidney function., (© 2023. The Author(s).)

Objective: Pregnancy is a state of physiological inflammation facilitating implantation. Early isolated hypothyroxinaemia (IH) and increased inflammation (including obesity) have been associated with severe obstetric complications. The current study evaluated the association between IH, low ferritin and inflammation parameters (interleukin 6 (IL-6), C-reactive protein (CRP), human chorionic gonadotrophin (hCG) and obesity. Moreover, the course of these parameters throughout pregnancy was evaluated in relation to IH., Methods: In the cross-sectional study (A) at 12 weeks, 2759 women participated and 2433 participated in the longitudinal study (B) with assessments at 12, 20 and 28 weeks gestation. At the first trimester, 122 (4.4%) IH women (free thyroxine (FT4) <5th percentile, normal TSH levels) were compared with 2114 (76.6%) reference women (FT4 between tenth and 90th percentiles, normal thyrotrophin (TSH) levels), in study B these figures were 99 (4.1%) and 1847 (75.9%), respectively., Results: Cross-sectionally, compared to reference women, IH was independently associated with low ferritin (<5th percentile, OR: 2.6, 95% CI: 1.4-4.9), high CRP (>95th percentile: OR: 1.9, 95% CI: 1.04-3.7), low hCG ( 30, OR: 1.7, 95% CI: 1.12.9) and higher age (OR: 1.1, 95% CI: 1.04-1.15). Longitudinally, compared to reference women, women with IH at 12 weeks gestation showed persistently and significantly lower ferritin and hCG levels, and persistently higher CRP and IL-6 levels throughout gestation., Conclusion: Gestational IH could be viewed as a condition of increased inflammation, as reported in non-thyroidal illness syndrome. Less favourable inflammation parameters and low iron status during early gestation in IH women seem to persist throughout gestation.

The Contrast Media Safety Committee of the European Society of Urogenital Radiology has, together with the Preanalytical Phase Working Group of the EFLM Science Committee, reviewed the literature and updated its recommendations to increase awareness and provide insight into these interferences., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: High plasma copper (Cu) and low zinc (Zn) levels have been associated with depression. However, most studies used low sample sizes and a cross-sectional design, and perinatal data are scarce. We investigated the possible association between pregnancy-specific psychological distress and the plasma CuZn ratio using a prospective design., Methods: Pregnancy-specific distress symptoms were assessed at each trimester by means of the Tilburg Pregnancy Distress Scale, negative affect subscale, in 2036 pregnant women. Cu and Zn were assessed at 12 wk of gestation in plasma samples by inductively coupled plasma mass spectrometry. Growth mixture modeling determined trajectories of women's pregnancy-specific negative affect (P-NA) symptoms, which were entered in a multiple logistic regression analysis as dependent variable and the CuZn ratio as independent variable., Results: Two P-NA symptom classes were found: 1) persistently low (n = 1820) and 2) persistently high (n = 216). A higher CuZn ratio was independently associated with persistently high P-NA symptom scores (odds ratio = 1.52; 95% confidence interval, 1.13-2.04) after adjustment for confounders. A sensitivity analysis was performed excluding all women with high P-NA scores at 12 wk of gestation (>1 SD above the mean P-NA score). In the 1719 remaining women, a higher CuZn ratio significantly predicted the development of increasing P-NA symptom scores after adjustment for confounders (odds ratio = 1.40; 95% confidence interval, 1.04-1.95)., Conclusions: A higher CuZn plasma ratio is an independent determinant of developing pregnancy-specific distress symptoms throughout pregnancy, suggesting that micronutrients could be used as novel biomarkers for psychological distress research of perinatal mood disorders., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: It is unclear whether levels of hypothyroid symptoms in pregnant women with (sub)clinical thyroid dysfunction differ from euthyroid controls and whether free thyroxine (fT4)/thyrotropin (TSH) changes throughout pregnancy affect hypothyroid symptom levels. The objective was twofold: (1) To compare hypothyroid symptom levels between thyroid dysfunction subgroups and a carefully defined reference group; (2) to assess the association between fT4/TSH changes throughout pregnancy and hypothyroid symptom levels adjusted for depressive symptoms. Methods: The current study was a longitudinal prospective cohort study in 1800 healthy pregnant women. At each trimester of pregnancy, hypothyroid symptoms were assessed with a 12-item symptom hypothyroidism checklist and depressive symptoms with the Edinburgh Depression Scale. Thyroid dysfunction was defined using the 2.5-97.5th fT4/TSH percentile of thyroid peroxidase antibodies-negative women. Euthyroid controls consisted of women with appropriate fT4 levels within the 10-90th percentile and with a normal TSH level. Hypothyroid symptom mean scores were compared between controls and several thyroid dysfunction subgroups. Growth mixture modeling was performed to evaluate possible longitudinal trajectories of hypothyroid and depressive symptoms. The association between hypothyroid symptom trajectories (adjusted for depression) and fT4/TSH changes was assessed with multivariate logistic regression analysis. Results: Women with overt hypothyroidism (fT4 < 2.5th, TSH >97.5th) and hypothyroxinemia (fT4 < 2.5th, TSH: 2.5-97.5th) showed higher hypothyroid symptom levels compared with the euthyroid controls and women with subclinical hypothyroidism (SCH, fT4: 2.5-97.5th, TSH >97.5th), because 82% of these SCH women had fT4 levels in the euthyroid range. Two groups of hypothyroid and depressive symptoms were defined: a persistently low and persistently high symptom group. fT4 decreased in 98% of the women from the first to third trimester and per unit pmol/L fT4 decrease (not TSH increase), the likelihood to present persistently high hypothyroid symptoms increased with 46%, adjusted for depression. Conclusions: A properly defined euthyroid control group distinguishes women with hypothyroid symptoms. An fT4 decrease toward end term is associated with persistently high hypothyroid symptom levels. Clinicians should be aware of the importance of fT4 stratification in SCH women.

Objectives: For the correct interpretation of test results, it is important to be aware of drug-laboratory test interactions (DLTIs). If DLTIs are not taken into account by clinicians, erroneous interpretation of test results may lead to a delayed or incorrect diagnosis, unnecessary diagnostic testing or therapy with possible harm for patients. A DLTI alert accompanying a laboratory test result could be a solution. The aim of this study was to test a multicentre proof of concept of an electronic clinical decision support system (CDSS) for real-time monitoring of DLTIs., Methods: CDSS was implemented in three Dutch hospitals. So-called 'clinical rules' were programmed to alert medical specialists for possible DLTIs based on laboratory test results outside the reference range in combination with prescribed drugs. A selection of interactions from the DLTI database of the Dutch society of clinical chemistry and laboratory medicine were integrated in 43 clinical rules, including 24 tests and 25 drugs. During the period of one month all generated DTLI alerts were registered in the laboratory information system., Results: Approximately 65 DLTI alerts per day were detected in each hospital. Most DLTI alerts were generated in patients from the internal medicine and intensive care departments. The most frequently reported DLTI alerts were potassium-proton pump inhibitors (16%), potassium-beta blockers (11%) and creatine kinase-statins (11%)., Conclusions: This study shows that it is possible to alert for potential DLTIs in real-time with a CDSS. The CDSS was successfully implemented in three hospitals. Further research must reveal its usefulness in clinical practice., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: Knowledge of possible drug-laboratory test interactions (DLTIs) is important for the interpretation of laboratory test results. Failure to recognize these interactions may lead to misinterpretation, a delayed or erroneous diagnosis, or unnecessary extra diagnostic tests or therapy, which may harm patients. The aim of this multicentre survey was to evaluate the clinical value of DLTI alerts., Methods: A survey was designed with six predefined clinical cases selected from the clinical laboratory practice with a potential DLTI. Physicians from several departments, including internal medicine, cardiology, intensive care, surgery and geriatrics in six participating hospitals were recruited to fill in the survey. The survey addressed their knowledge of DLTIs, motivation to receive an alert and opinion on the potential influence on medical decision making., Results: A total of 210 physicians completed the survey. Of these respondents 93% had a positive attitude towards receiving DLTI alerts; however, the reported value differed per case and per respondent's background. In each clinical case, medical decision making was influenced as a consequence of the reported DLTI message (ranging from 3 to 45% of respondents per case)., Conclusions: In this multicentre survey, most physicians stated DLTI messages to be useful in laboratory test interpretation. Medical decision making was influenced by reporting DLTI alerts in each case. Alerts should be adjusted according to the needs and preferences of the receiving physicians., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Physicians are often guided by laboratory values. When a clinical presentation does not match laboratory values, one must consider the possibility that these values may be falsely increased or decreased. A common cause is analytical interference., Case Description: A 57-year-old male, presenting with fatigue and palpitations, had high TSH and normal FT4 values. Although there were no fitting clinical symptoms for these values, the patient was treated with levothyroxine assuming he had subclinical hypothyroidism. TSH levels remained high, however, whereas FT4 levels increased and the patient developed thyrotoxicosis. Eventually, it was discovered that the TSH was falsely elevated., Conclusion: The patient turned out to have macro TSH, where TSH forms conjunctions with IgG into larger molecules. These conjugates cause a rarely occurring interference during laboratory analysis, resulting in a falsely increased TSH value.

Background: The analysis of insulin-like growth factor I (IGF-I) is an important tool for pediatricians in the diagnosis and treatment of growth hormone deficiency in children. However, significant differences exist in IGF-I assays and normative datasets, which can have important clinical consequences., Methods: IGF-I analyses were performed using the IDS-iSYS platform on 1,897 samples from pediatric patients (0.5-18 years old). Z-scores were calculated based on normative IGF-I data from Bidlingmaier et al. (SD-BM) [J Clin Endocrinol Metab. 2014 May; 99(5): 1712-21] and normative IGF-I data from the IGF-I harmonization program in the Netherlands (SD-NL). The differences in Z-scores were analyzed at relevant clinical decision points (-2 SD, +2 SD). These normative datasets were also compared to normative data reported by Elmlinger et al. [Clin Chem Lab Med. 2004; 42(6): 654-64]., Results: The difference in Z-score between SD-BM and SD-NL was highest in males between 0 and 3 years old, exceeding 2 SD. Clinically relevant discordance between both Z-scores at -2 and +2 SD was found in 12.7% of all samples. The IGF-I levels at -2 and +2 SD reported in the normative dataset of Elmlinger et al. were up to 100% higher than the IGF-I levels reported by Bidlingmaier et al. or the Dutch harmonization program., Conclusion: Pediatricians and laboratory specialists should be aware of relevant differences that can exist between IGF-I assays and normative data. Well-defined pediatric reference ranges for the IDS-iSYS platform are highly desirable., (© 2018 S. Karger AG, Basel.)

Background: The variety of LC-MS/MS methods measuring total 25(OH)D used today is vast and the comparability among these methods is still not well assessed., Methods: Here, we performed a comparison in samples of healthy donors between the currently routinely used 25(OH)D LC-MS/MS methods in the Netherlands and the Ghent University reference measurement procedure to address this issue (n=40). Additionally, an interlaboratory comparison in patient serum samples assessed agreement between the Dutch diagnostic methods (n=37)., Results: The overall correlation of the routine methods for 25(OH)D 3 with the reference measurement procedures and with the mean of all diagnostic methods was excellent (r>0.993 and r>0.989, respectively). Three out of five methods aligned perfectly with both the reference measurement procedure and the median of all methods. One of the routine methods showed a small positive bias, while another showed a small negative bias consistently in both comparisons., Conclusion: The biases most probably originated from differences in calibration procedure and may be obviated by reassessing calibration of stock standards and/or calibrator matrices. In conclusion, five diagnostic centers have performed a comparison with the 25(OH)D Ghent University reference measurement procedure in healthy donor serum samples and a comparison among themselves in patient serum samples. Both analyses showed a high correlation and specificity of the routine LC-MS/MS methods, yet did reveal some small standardization issues that could not be traced back to the technical details of the different methods. Hence, this study indicates various calibration procedures can result in perfect alignment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC-MS/MS methods is available. We compared eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione., Methods: Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC-MS/MS methods. We performed Passing-Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated., Results: The concentrations ranged from 0.05-1.26 nmol/L, 6.15-24.44 nmol/L and 0.15-4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7-16.0%, 0.9-5.2% and 1.2-9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90-1.25, 0.87-1.24 and 0.94-1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods., Conclusion: Although most routine LC-MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved., (Copyright © 2016. Published by Elsevier B.V.)

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease, characterized by microangiopathic hemolytic anaemia and thrombocytopenia, resulting in neurologic and/or renal abnormalities. We report a 49-year-old patient with a history of thrombotic events, renal failure, and thrombocytopenia. Blood analysis demonstrated no ADAMTS13 activity in the absence of antibodies against ADAMTS13. The complete ADAMTS13 gene was sequenced, and two mutations were identified: one mutation on exon 24 (Arg1060Asp), which had previously been described, and a mutation on exon 27 (Met1260IlefsX34), which has not been reported. For these mutations, compound heterozygosity appears to be necessary to cause TTP, as family members of the patient display only one of the mutations and all displayed normal ADAMTS13 activity.

Objective: To describe the consequences on the burden for primary and secondary care in the Netherlands, of using age-related cut-off values for renal function which follow the Dutch national transmural agreement (LTA) for 'Chronic renal impairment', rather than the 'Kidney disease outcome quality initiative' (K/DOQI) guidelines., Design: Observational cross-sectional study., Methods: 82,424 patients whose serum creatinine had been determined in 2009 were identified from the laboratory registry of the Isala Clinics in Zwolle, the Netherlands. The glomerular filtration rate was estimated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation (eGFR). Burden of care was defined as the necessity for referral or consultation in secondary care. The number of people that would have been referred using the K/DOQI guideline that refers all those with an eGFR < 60 ml/min/1.73 m², was compared to a situation using age-related cut-off values in the referral policy., Results: The study population contained 82,424 people; 45.3% were men; age range was 19-106 years; 38.7% were > 65 years. 19% of the population (n = 15,637) had an eGFR < 60 ml/min/1.73 m2 and would have been referred had the K/DOQI guidelines been applied; 11,935 of those 15,637 were > 65 years. The use of the LTA for 'Chronic renal impairment', that includes age as one of the criteria, would have resulted in the referral of 3,303/15,637 patients (2,011 of those 3,303 were > 65 years), and resulted in consultation with a nephrologist for 5,748/15,637 patients (3,338/5,748 were > 65 years). The majority of patients aged > 65 years and with an eGFR < 60 ml/min/1.73 m2 (55%) could be treated in primary care without consultation of secondary care or referral., Conclusion: The categorization applied by the current LTA for 'Chronic renal impairment', whereby age-related cut-off values are used in the referral policy, will result in more targeted referral to secondary care, especially in the elderly patient group, when compared to application of the K/DOQI guidelines.

Turbulent-flow chromatography (TFC) on-line coupled to liquid chromatography mass spectrometry (LC-MS) is used to determine flavonoids and resveratrol in different types of wines. A fully automated system was developed in which 10 mL of sample (diluted wine) was passed over a TFC column, after which the retained analytes were separated by reversed-phase LC and detected by negative ion mode atmospheric-pressure chemical ionization (APCI) MS. The method proved to be fast, non-laborious, robust and sensitive. The feasibility of the method was tested on several red, white and rose wines. Quantitation of resveratrol was possible using the standard addition procedure. Red wine showed the highest amount of resveratrol (4 mg L(-1)), while rose and white wine contained concentrations which were about ten fold lower.

The online, selective isolation of protein-ligand complexes using cobalt(II)-coated paramagnetic affinity beads (PABs) and subsequent liquid chromatography-mass spectrometry (LC-MS) determination of specifically bound ligands is described. After in-solution incubation of an analyte mixture with His-tagged target proteins, protein-analyte complexes are mixed with the Co(II)-PABs and subsequently injected into an in-house built magnetic trapping device. Bioactive ligands bound to the protein-Co(II)-PABs are retained in the magnetic field of the trapping device while inactive compounds are removed by washing with a pH 7.4 buffer. Active ligands are online eluted toward the LC-MS system using a pH shift. In the final step of the procedure, the protein-Co(II)-PABs are flushed to waste by temporarily lowering the magnetic field. The proof-of-principle is demonstrated by using commercially available Co(II)-PABs in combination with the His-tagged human estrogen-receptor ligand-binding domain. The system is characterized with a number of estrogenic ligands and nonbinding pharmaceutical compounds. The affinities of the test compounds varied from the high micromolar to the subnanomolar range. Typical detection limits are in the range from 20 to 80 nmol/L. The system is able to identify binders in mixtures of compounds, with an analysis time of 9.5 min per mixture. The standard deviation over 24 h is 9%.

A novel methodology is shown enabling the screening of mixtures of compounds for their affinity to a receptor protein. The system presented, dynamic protein-affinity chromatography solid-phase extraction (DPAC-SPE), overcomes the limitations of the existing methods by performing an incubation of the His-tagged protein with a mixture of possible ligands, which are still in their native conditions. This is followed by a fully automated affinity trapping step, coupled on-line to an LC-MS system in order to detect and identify the bound ligands. The system has been optimized using a commercially available on-line SPE system, using the estrogen receptor alpha (ERalpha) as model protein. A representative range of ligands with sub-nanomolar to millimolar affinities has been identified successfully from a mixture. The weakest binder that can be identified is norethindrone (approximately K(d)=0.1-1mM). The same setup also provides the possibilities to measure EC50 curves of both weak and strong binders.

An integrated online screening system was developed to rapidly screen homogeneous catalysts for activity toward a selected synthesis. The continuous-flow system comprises standard HPLC pumps for the delivery of substrates, an HPLC autosampler for the injection of homogeneous catalysts, a thermostated reactor to mediate synthesis, and a single-stage quadrupole mass spectrometer (MS) equipped with atmospheric pressure chemical ionization for the determination of product formation. MS detection offers sensitivity, specificity, and speed when applied to the analysis of dynamic processes in the condensed phase. By applying the present methodology for the study of substrate conversion mediated by homogeneous catalysts, the concentration of substrates and reaction product could be monitored while information about the catalysts could also be obtained. In an initial screening application, the performance of a selected number of Lewis acids in the multicomponent synthesis of a highly substituted 2-imidazoline was determined. Limit of detection and limit of quantitation were determined by injecting different concentrations of 2-imidazoline standards and proved to be 1.6 and 5.2 nM, respectively. The results obtained with the new screening method were in good agreement with a traditional bench-scale experiment. Moreover, the system was capable of determining catalyst performance with very low catalyst and solvent consumption while the ruggedness of the system was exhibited with a 24-h continuous analysis of 280 successive catalyst injections with a peak area variation within 7% relative standard deviation.

Electrospray ionization mass spectrometry was used to investigate complex formation of different metal complexes in a continuous-flow ligand-exchange reactor. A computer program was developed based on normal equilibrium calculations to predict the formation of various metal-ligand complexes. Corresponding to these calculations, infusion electrospray mass spectrometric experiments were performed to investigate the actual complex formation in solution. The data clearly show good correlation between the theoretically calculated formation of metal-ligand complexes and the experimental mass spectrometric data. Moreover, the approach demonstrates that the influence of the pH can be investigated using a similar approach. Indirectly, these infusion experiments provide information on relative binding constants of different ligands towards a metal-ion. To demonstrate this, a continuous-flow ligand-exchange detection system with mass spectrometric detection was developed. Injection of ligands, with different affinity for the metal-ion, into the reactor shows good correlation between binding constants and the response in the ligand-exchange detection system. Additional information on the introduced ligand, and the complexes formed after introduction of the ligand, can be obtained from interpretation of the mass spectra.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.